The Mosquito Control District set free 20,000 male Aedes aeqgypti mosquitoes, after company in Kentucky manually injected each one with Wolbachia bacteria.
When the infected male mosquitoes mate with females, the eggs they produce will not hatch.
It is hoped the 12 week field trial on Stock Island, just north of Key West, will reduce or eliminate the population of female mosquitoes, which bite humans transmitting diseases like Dengue fever and Chikungunya, as well as Zika.
"The eggs never even hatch," said Stephen Dobson, founder of Mosquitomate, which injected the creatures.
The infected mosquitoes were flown in cardboard tubes — similar to ones used in paper towel rolls — from Lexington, Kentucky, to Key West.
At the 25 acre test site district staff released them by shaking or blowing into the tubes, said Andrea Leal, the district's executive director.
"They liked the humidity," Ms Leal said. "They were very happy mosquitoes."
The trial is expected to last about three months, with twice-weekly releases. Seven Wolbachia-infected males should be released for every one wild male to drive down the mosquito population, Mr Dobson said.
To date, 5,238 cases of Zika virus have been reported in the continental US and Hawaii, according to the Centre for Disease Control and Prevention (CDC).
Less than 300 reported cases were locally transmitted and in the continental US all of those occurred in Florida and Texas. Most people were infected while travelling to areas where the virus is more prevalent.
One in 10 Zika-infected mothers had babies with related birth defects in the United States last year, according to the CDC.
“A successful trial with the Wolbachia-infected mosquitoes could mean the availability of a new tool in the fight against the Aedes aegypti mosquito for not only our District, but for Mosquito Control Districts around the country,” Ms Leal told CNN .
"We're looking at these sterile insect techniques because our conventional mosquito control methods are costly and labour-intensive," she added.
Stock Island is about 130 miles southwest of Miami, where Aedes aegypti mosquitoes were blamed for spreading the Zika virus last year.
Battling the zika virus - in pictures
Battling the zika virus - in pictures
A worker of the Salvadorean Ministry of Health fumigates a house in Soyapango, 6 kilometers from San Salvador, El Salvador. Salvadorean authorities have began a three days campaign of fumigation to reduce the presence of the mosquito that transmit the Zika virus.
A Health Ministry employee fumigates a home against the Aedes aegypti mosquito to prevent the spread of the Zika virus in Soyapango, six km east of San Salvador. Health authorities have issued a national alert against the Aedes Aegypti mosquito, because of the link between the Zika virus and microcephaly and Guillain-BarrÈ Syndrome in foetuses.
AFP PHOTO/Marvin RECINOSMarvin RECINOS/AFP/Getty Images
A pediatric infectologist examines a two-months-old baby, who has microcephaly, on 26 January 2016 in Recife, Brazil.
A woman walks through the fumes as Health Ministry employee fumigate against the Aedes aegypti mosquito to prevent the spread of the Zika virus in Soyapango.
Marvin RECINOS/AFP/Getty Images
A health ministry employee sprays to eliminate breeding sites of the Aedes Aegypti mosquito, which transmits diseases such as the dengue, chicunguna and Zica viruses, in a Tegucigalpa cemetery on January 21, 2016. The medical school at the National Autonomous University of Honduras (UNAH) recommended that women in the country avoid getting pregnant for the time being due to the presence of the Zika virus. If a pregnant woman is infected by the virus, the baby could be born with microcephaly.
AFP PHOTO/Orlando SIERRA
A man walks away from his home with his son as health workers fumigates the Altos del Cerro neighbourhood as part of preventive measures against the Zika virus and other mosquito-borne diseases in Soyapango, El Salvador
A three-months-old, who has microcephaly, in Recife, Brazil.
A pregnant woman waits to be attended at the Maternal and Children's Hospital in Tegucigalpa. The medical school at the National Autonomous University of Honduras (UNAH) recommended that women in the country avoid getting pregnant for the time being due to the presence of the Zika virus. If a pregnant woman is infected by the virus, the baby could be born with microcephaly.
ORLANDO SIERRA/AFP/Getty Images
Army soldiers apply insect repellent as they prepare for a clean up operation against the Aedes aegypti mosquito, which is a vector for transmitting the Zika virus in Sao Paulo, Brazil.
AP Photo/Andre Penner
Workers disinfect the Sambadrome in Rio de Janeiro to fight the spread of the Zika virus
Dr. Vanessa Van Der Linden, the neuro-pediatrician who first recognized the microcephaly crisis in Brazil, measures the head of a 2-month-old baby with microcephaly in Recife
Mother Mylene Helena Ferreira cares for her son David Henrique Ferreira, 5 months, who has microcephaly, on January 25, 2016 in Recife, Brazil. In the last four months, authorities have recorded close to 4,000 cases in Brazil in which the mosquito-borne Zika virus may have led to microcephaly in infants
U.S. women who are pregnant from traveling to many South American countries
In the last four months, authorities have recorded close to 4,000 cases in Brazil in which the mosquito-borne Zika virus may have led to microcephaly in infants.
Dr. Vanessa Van Der Linden, the neuro-pediatrician who first recognized the microcephaly crisis in Brazil, examines a two-month-old baby with microcephaly on January 27, 2016 in Recife, Brazil
Brazil is one of the countries in South America where the Zika virus has taken hold
Health workers fumigating to combat Zika virus in Lima, Peru. The US have already issued a warning urging pregnant women to avoid travel to Latin American countries
Two-month-old Jose Wesley, born with microcephaly in Brazil, is nursed by his brother
Officials are still considering a separate test of mosquitoes genetically modified by the British biotech firm Oxitec to produce Aedes aegypti offspring that die outside a lab.
The US Food and Drug Administration initially approved a trial in a residential neighbourhood near Key West, saying the test would not significantly affect the environment, but outrage from anti-GMO activists and residents forced the district to find a new location.
The FDA is currently seeking public comment on a proposal clarifying which mosquito-related products it regulates and which ones would be regulated by the EPA, which has stalled progress.
Mr Dobson said he hopes the regulatory issues will be resolved quickly, so that mosquito controllers will have more tools to keep the viruses carried by invasive mosquito species from spreading, and more flexibility to address the public's concerns about human health.
"It might be in some places that people will say, 'We don't want Wolbachia,' or they'll say, 'We don't want GMO,' or they'll say, 'We don't want planes spraying overhead,'' Mr Dobson said.
Additional reporting by Associated PressReuse content