Northampton flanker Calum Clark has been ruled out of England action for a third time in 12 months after he failed to overcome an ankle injury in time to make the tour of Uruguay and Argentina.
England have also lost Leicester lock Ed Slater from their touring party after he suffered a recurrence of a triceps injury in the Tigers' Aviva Premiership final victory over Northampton.
Wasps-bound lock Kearnan Myall is the only player to have been called up as a replacement, with England now taking 31 players for Sunday's tour match in Montevideo and the two Tests against the Pumas.
For Clark, 23, it is yet another international set-back.
Clark was ruled out of England's 2012 summer tour of South Africa and the autumn internationals that followed after receiving a record 32-week ban for breaking the arm of Leicester of hooker Rob Hawkins.
England head coach Stuart Lancaster stood by Clark and he was recalled for the RBS 6 Nations, only to suffer a shoulder injury in January that sidelined him for the whole tournament.
Clark was selected again for England's three-match tour of South America but he failed a fitness test with Northampton on Thursday and will not be ready for the forthcoming Test series.
"I feel for him. He is desperately unlucky, one to miss out on a big final for his club - he played well in the semi-final - and to miss out on a tour again compounds the frustration from missing the Six Nations," Lancaster said.
"He has been unlucky with the injuries. I had a good chat with him yesterday - he came in and we assessed his ankle and it clearly was not going to be right until beyond the first Test and as a consequence it would take him out of contention.
"It makes sense to leave him here and get the ankle right, have a good pre-season and push forward to next season.
"He was disappointed. He was quite resigned to it - he has had it for two weeks and having failed the fitness test on Thursday for Northampton he realised that it was going to be a long shot to get on the tour."
Lancaster decided not to replace both Clark and Slater because he feels England have sufficient options in both the second row and back row.
England play a combined South American XV on Sunday in Montevideo and then Tests against Argentina in Salta on June 8 and Buenos Aires on June 15.
Lancaster plans wholesale changes for next weekend, with the Northampton contingent who tasted bitter defeat to Leicester in that final all set to be given a chance to "get straight back on the horse".
Stephen Myler, Luther Burrell, Courtney Lawes, Lee Dickson, Ben Foden and England captain Tom Wood have all been pencilled in to start in Montevideo.
"My job is to pick them up, give them a clear target going into next Sunday and get some of them motivated to play again," Lancaster said.
"The likes of Courtney Lawes, Tom Wood, Lee Dickson, I thought they put in an unbelievable shift and I want to make sure they come back to England and get back on the horse.
"The main thing for me this week is to rotate squad and give as many players an opportunity as I can leading up to the first Test.
"There will be a significant number of changes from the Barbarians game with a few players who played in the Barbarians game rested going into the two Tests."