To elucidate whether breast milk, vaginal discharge and contamination with maternal blood at birth are possible routes of mother-to-child transmission of hepatitis C virus (HCV), we examined HCV RNA in the cord and peripheral blood of infants, and in the blood, vaginal discharge, and breast milk of anti-HCV seropositive mothers. From July 1991 to July 1992, we studied 20 healthy pregnant women, who were seropositive with the Ortho anti-HCV EIA, and their infants. Using a sensitive nested polymerase chain reaction (nested PCR), we investigated the presence or absence of hepatitis C virus in the above-mentioned specimens. Moderate elevation of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) was observed in only one woman in the first and third trimesters. The nested PCR and subsequent Southern hybridization detected 0.5-5.5 copies of HCV c-DNA. HCV RNA was detected in 17/20 blood samples (85%), 7/14 vaginal discharge samples (50%) and 4/10 cord blood samples (40%). However, no HCV RNA was identified in the peripheral blood of infants or breast milk. The mother-to-child transmission of HCV at delivery or via breast milk does not appear to contribute much to maintaining the global HCV reservoir.