Barcelona and Real Madrid were mired in fresh controversy last night after Pepe appeared to stamp on the hand of Lionel Messi during a 2-1 home defeat for José Mourinho's team.

The incident, in the 68th minute of their Copa del Rey quarter-final first leg, is the latest to scar the meetings between the two teams. Episodes over the past year include Mourinho poking a Barcelona coach in the eye, a sending-off for Pepe in last season's Champions League semi-final, extraordinary outbursts from both managers and allegations of racism, dirty tricks and diving.

Messi was on the ground after being fouled by José Callejón when Pepe appeared to deliberately tread on the left hand of the world player of the year. The referee booked Callejón but took no action against Pepe, who was on a yellow card.

Both managers claimed not to have witnessed the flashpoint and the Spanish football federation will be able to take retrospective action only if a mention is included in the referee's report.

"I didn't see it but of course if it happened it would be worthy of censure," said Mourinho. His opposite number, Pep Guardiola, said: "The players told me about it but I haven't seen it on the television. You lot saw it on television and you have to decide what you think."

The Manchester United striker Wayne Rooney, who had been following the match on television, tweeted: "Pepe. What an idiot."

That drew a response from the retired former Real Madrid player Guti, who wrote: "I laugh at Rooney. There are plenty of saints – in heaven. If he wants to be a saint, he should set an example. Making mistakes is human."

Goals from Carles Puyol and Eric Abidal won the match for Barcelona after Cristiano Ronaldo had given Madrid the lead. Mourinho was angry at the ease with which Puyol equalised with a header from a corner.

"We had not even sat down on the bench when that went in," Mourinho said. "You cannot concede a goal like that from a dead ball when you are 1-0 up."

This was Barcelona's ninth victory in 13 matches against their arch rivals. "To come and win so often is not normal," said Guardiola. Mourinho accepted the blame. "Victory has many fathers, defeat has just one – and that's me," he said.