The head of the Vatican Bank, Ettore Gotti Tedeschi, is under investigation as part of a money-laundering inquiry, police sources say.
Prosecutors also seized 23m euros ($30m; £19m) from the bank’s accounts with another smaller institution.
The inquiry was launched after two suspicious transactions were reported to tax police in Rome.
The Vatican said it was „perplexed and astonished“, and expressed full confidence in Mr Tedeschi.
In a statement, the Vatican said it had been working for some time to make its finances more transparent to comply with anti-terrorism and anti-money laundering regulations.
Investigators are looking into claims that Mr Tedeschi and another senior official violated laws that require banks to disclose information on financial operations.
The BBC’s David Willey in Rome says the Bank of Italy’s financial intelligence unit tipped off Italy’s tax police last week, after two suspicious transactions were reported between the Vatican Bank and two different Italian banks.
The tax police seized one deposit of 23m euros that the Vatican Bank had deposited with a small Italian bank called Credito Artigianato, our correspondent says.