The Vatican bank's top two officials are under investigation for suspected money laundering.
Italian judicial sources say police have also frozen 23 million euros (£19.4 million) of its funds.
They said President Ettore Gotti Tedeschi and director-general Paolo Cipriani were being investigated by Rome magistrates in a case involving alleged violations of European Union money-laundering rules.
The Vatican confirmed the Rome magistrates' action in a statement that expressed "perplexity and amazement" at the move and "utmost faith" in the two men who head the bank, officially known as Institute for Religious Works (IOR).
Gotti Tedeschi, 65, has been at the helm of the bank for a year and is a close adviser to Italian Economy Minister Giulio Tremonti.
The sources said Italy's financial police had preventively frozen 23 million euros of the IOR's funds in an account in an Italian bank in Rome.
Two recent transfers from an IOR account in the Italian bank were deemed suspicious by financial police and blocked.
A statement from the Vatican's Secretariat of State said the bank had committed no wrongdoing because it was transferring its own money between its own accounts.
Gotti Tedeschi, a devout Catholic who has taught financial ethics at the Catholic University of Milan, also heads an Italian unit of the Spanish Banco Santander, as well as serving on the board of several major Italian banks.
The IOR primarily manages funds for the Vatican and religious institutions around the world, such as charity organisations and religious orders of priests and nuns.
Its cash point machines in the Vatican are perhaps the only ones in the world that allow clients to choose Latin as the language to perform operations.
The IOR was involved in the fraudulent bankruptcy of Banco Ambrosiano, then Italy's largest private bank, that turned into an international scandal in 1982. The IOR held a small stake in Ambrosiano, whose president Roberto Calvi was found hanged under London's Blackfriars Bridge the same year.
Several investigations failed to determine whether Calvi, known as God's Banker, had been killed or committed suicide.
The Vatican denied any responsibility for the collapse of Banco Ambrosiano but made what it called a "goodwill payment" of $250 million (£161 million) to creditors.