Several studies have demonstrated that hepatitis C virus (HCV) may be transmitted from mother to offspring. To date, however, little is known about the risk of vertical transmission during subsequent pregnancies. The purpose of this study was to evaluate the risk of vertical HCV transmission in offspring in subsequent pregnancies of HCV infected women. In a multicenter study, two groups of index cases were selected. Group 1 included 75 children investigated for HCV infection during prospective studies of vertical transmission. Group 2 included children born to HCV-infected mothers and found to be HCV infected, independent of studies on vertical transmission. All children in the index cases had one or more siblings. Anti-HCV, HCV-RNA (determined by polymerase chain reaction), and HCV genotype were evaluated in all the infected children, their mothers, and siblings. The results indicate that a mother who has already delivered an HCV-infected baby is not at greater risk of infecting her second child. Duration of maternal infection does not seem to be a risk factor in offspring infection, because HCV infection is equally distributed among first-born infants and infants of subsequent births. Because clustering of HCV infection among siblings appeared to be rare in this study, data also indirectly confirm that the risk of horizontal transmission of HCV among siblings is low. For practical purposes, the current observations indicate that mothers who have already delivered an HCV-infected child can be advised that this event does not increase the probability of infecting the second child.