Hepatitis A virus (HAV) infection is endemic in Puglia (South Italy). Epidemiological studies indicate that shellfish consumption, particularly mussels, is a major risk factor for HAV infection, since these products are eaten raw or slightly cooked. Nested reverse transcriptase-polymerase chain reaction (RT-PCR) has been shown to be a sensitive technique for the detection of HAV in mussels. The aim of the present study was to detect the presence of HAV in a large sample of mussels by nested RT-PCR and to confirm the presence of infectious viral particles in positive samples by cell culture infection and RT-PCR confirmation. Two hundred and ninety samples of mussels from different sources were collected between December 1999 and January 2000. One hundred samples were collected before being subjected to depuration, 90 after depuration, and 100 were sampled in different seafood markets. HAV-RNA was detected in 20 (20.0%) of non-depurated mussels, in 10 (11.1%) of depurated samples, and in 23 (23.0%) of samples collected in the shellfish markets, without any significant difference in the prevalence of positive samples by collection sources (chi2 = 4.79, p = 0.09). Of the 53 samples found positive by nested RT-PCR, 18 (34.0%) resulted positive by cell culture assay. No relationship between viral contamination and bacterial contamination was found (p = 0.41). This study confirms the usefulness of molecular techniques in detecting HAV in shellfish and, thus, for the screening of a large sample of naturally contaminated mussels. Improved shellfish depuration methods are needed to obtain virus-safe shellfish and reduce the risk for public human health.