The risk of mother-to-infant transmission of hepatitis C virus (HCV) varies according to the population studied and the tests used. Aim of the current study was to investigate HCV vertical transmission rate in children born to 30 HCV positive/HIV negative pregnant women in Italy. We investigated the potential vertical transmission of HCV by identifying HCV antibody seropositive pregnant women, by analyzing HCV-RNA in the peripheral blood using PCR and by prospectively following their offspring until 24 months of age. During the third trimester, 2,980 consecutive pregnant women were examined for anti-HCV antibodies by a second generation Enzyme-Linked Immunosorbent Assay (EIA2) and re-assayed by a second generation Recombinant Immunoblot Assay (RIBA2). A total of 32 mothers (1.07%) were positive for EIA2 test; 30 out of 32 had a reactive confirmatory RIBA2 test for HCV. All anti-HCV positive mothers were negative for HIV. These 30 mothers and their 30 babies formed the study cohort. Of the 30 anti-HCV positive mothers, 10 were also positive for serum HCV-RNA by PCR. All the babies born to the 30 anti-HCV positive mothers were initially negative for HCV-RNA (cord blood specimens), but three babies became positive at three months of age and remained positive thereafter. These babies had been born to 3 of the 10 mothers with viremia during the third trimester of pregnancy, These results suggest that HCV vertical transmission is possible in 10% of anti-HCV positives and in about 33% of the HCV-RNA seropositive mothers.