Many in Croatia regard Gen Gotovina as a national hero
Carla del Ponte, chief prosecutor for war crimes in the former Yugoslavia, has said she believes Gen Ante Gotovina is hiding in a monastery in Croatia.
Ms del Ponte's spokeswoman told the BBC News website that the Vatican had refused to help in the search for him, despite being in a position to do so.
A spokesman for the Croatian Catholic Church rejected the charges.
In a statement later the Vatican said that Archbishop Giovanni Lajolo, responsible for the city-state's foreign relations, had asked Ms del Ponte for further details of her assertions.
Chief spokesman Joaquin Navarro-Valls said Ms del Ponte had not yet responded to requests for further information.
Divided opinions
Gen Gotovina has been charged with the deaths of 150 Serb civilians in 1995.
Earlier this year, the European Union cited Zagreb's failure to arrest him as the reason behind delaying talks on Croatia's entry into the bloc.
Forces under Gen Gotovina's command are accused of killing scores of Serbs and expelling up to 200,000 from the Krajina region, now part of Croatia.
Many in Croatia regard him as a national hero.
Ms del Ponte wants the authorities to act against war criminals
The Croatian Bishops' Conference, which heads the Croatian Roman Catholic Church, dismissed Ms del Ponte's allegations.
Its spokesman Antun Suljic said the conference "has no knowledge or indications of the whereabouts" of Gen Gotovina.
Vatican 'refusal'
Ms del Ponte wrote to Pope Benedict XVI in July this year in an effort to secure the Vatican's co-operation, her spokeswoman told the BBC News website.
The Pope has yet to reply to the prosecutor's request for a meeting, the spokeswoman said.
Ms del Ponte earlier told British newspaper The Daily Telegraph she believed Gen Gotovina was "hiding in a Franciscan monastery and so the Catholic Church is protecting him".
"I have taken this up with the Vatican and the Vatican totally refuses to co-operate with us," she said.
Archbishop Lajolo told Ms del Ponte the Vatican did not know of Gen Gotovina's whereabouts and was not obliged to help her, she was quoted as saying.
Her spokeswoman, Florence Hartmann, told the BBC News website that "the law applies to everyone, including the Vatican".
She said UN prosecutors faced similar difficulties in tracking down the Bosnian Serb war crimes suspect Radovan Karadzic, who is thought to have sought refuge among Orthodox Christian monks in Montenegro.
"We're fully ecumenical," she said.
United Nations Chief War Crimes prosecutor, Carla Del Ponte, prosecutor for the International Criminal Tribunal for the former Yugoslavia has accused the Catholic Church in Croatia of shielding one of the most wanted war criminals of the conflict in the region.

The Vatican has denied the accusation that it is shielding the alleged war criminal, General Ante Gotovina, claiming they have sought clarification on Ms del Ponte's accusation that the General is being sheltered in a monastery in Croatia.