Alex Song is to miss the second leg of Arsenal's UEFA Champions League quarter-final against Barcelona on Tuesday night.
Song suffered a groin injury in the Premier League victory over Wolves at the weekend and had to be taken off with almost 20 minutes remaining.
Wenger later expressed hope that Song would be fit for European duty but admitted he would have to be assessed nearer to kick-off.
But it has now been confirmed that the Cameroon international will not feature at Camp Nou after his name was left off the 18-man squad travelling to Spain.
The 22-year-old will be sidelined with a fresh knee problem and Wenger admits it is a major blow.
"It was a bad surprise this morning. He had a scan yesterday afternoon on his knee and it is inflamed cartilage," said Wenger.
"We will have to wait and see how serious it is but he is completely out of the squad for tomorrow's game."
Song has been an important player in midfield this season but was expected to play in central defence against Barca in the absence of William Gallas.
Gallas may not play again this season due to a calf strain and Wenger is now likely to turn to veteran Sol Campbell alongside Thomas Vermaelen.
Wenger's preparations for the second leg have been hit by several injuries to key players, with Cesc Fabregas and Andrey Arshavin joining Song and Gallas on the sidelines.
Robin van Persie (ankle) is back in training, but is still a couple of weeks away from a return.
Wenger will not therefore be able to send out a full-strength team as the Gunners look to make it through to the last four.
The tie is at least delicately poised after a pulsating first leg at Emirates Stadium when Arsenal recovered from an initial onslaught to draw 2-2.