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.
The Vatican Bank, known officially as the Institute for Religious Works (IOR), was created during World War II to administer accounts held by religious orders, cardinals, bishops and priests.