The move came after Darren Bent limped out of training on the eve of the match. The Spurs forward had himself been a late call-up to the squad after Emile Heskey and Carlton Cole suffered injuries in Saturday's 4-0 friendly win over Slovakia, but his involvement with the national squad lasted only a few minutes before he was forced to head for the treatment room.
Agbonlahor has been added to the squad as cover with head coach Fabio Capello indicating that he plans to start the match with Peter Crouch leading the attack. The Villa forward's call-up represents a fresh snub to Michael Owen, although Capello again insisted that the injury-plagued Newcastle forward's England career could be revived once he starts playing regularly for his club.
"In every interview I've said the door for all the England players is open," Capello said. "I respect Owen but I chose Agbonlahor because he has played a lot of games."
Rio Ferdinand, who returned to training after missing the Slovakia match with a back strain, is set to start alongside John Terry in central defence. Capello will also have Shaun Wright-Phillips, who missed Saturday's game with an ankle injury, available.
With four straight wins in Group 6, England will be in a very strong position to reach next year's finals in South Africa if they can beat Ukraine, the only team they have yet to face. The win over Slovakia augured well for England's chances and Capello expects Ukraine to play a similar style.
"It will be a very dangerous game, they play very well on the counter attack," the Italian said. "It will be very tough. I was very happy after the Slovakia match, our movement with and without the ball was very good and we created a lot of good chances. For me it was a good test."
Capello believes Ukraine's tendency to lay back and try and hit teams on the counter makes the match ideally suited for Crouch. "I think the most important thing is the movement of Gerrard, Lennon or Beckham and Rooney," he said. "The movement near Crouch is very important.
"He can do the same movement and press like (Emile) Heskey. But the movement of the players around him will be very important. All the players will be important. We have to play like a team, a group, this is the most important thing. Not a single player."
Capello appears to have worked out how to get the best out of Rooney with the Manchester United forward having scored seven times in his last four internationals.
Rooney will reach 50 caps against the Ukraine and Ferdinand underlined how central a figure his Manchester United team-mate had become.
"He's beginning to play consistently well for England," Ferdinand said. "He's improving in the right way. Obviously I wasn't involved at the weekend and I could watch from the side and get a better look at how individuals were playing.
"Wayne was probably our best player by quite a distance on Saturday. He was doing the things that the manager asked of him. He picked the ball up in dangerous areas, put people in on goal and got two goals as well. He's doing what the manager wants to get out of the team."