Southampton boss Ronald Koeman says midfielder Jordy Clasie and defender Steven Caulker will miss Saturday's Premier League match at home to Everton.
And new boy Clasie, who signed from Feyenoord in the summer, could be out for three weeks after he was injured while playing for the U21 side on Monday night.
Koeman said: "It's a pity, because we have some injuries already in the beginning of the season.
"Jordy Clasie was back from his hamstring injury and he needed to play 60 minutes for the U21s against Manchester City on Monday, but he now has an ankle ligament injury. He will be out for two or three weeks.
"We still have Ryan Bertrand not available, and of course Fraser Forster and Florin Gardos we know about.
"Also, Steven Caulker is not available for the weekend. He has picked up a sinus infection and he had some medication.
"He's not in a good physical shape for this weekend, but we hope he will return for Thursday (Europa League tie against FC Midtjylland)."