To assess the seroepidemiology of hepatitis C virus (HCV) infection in pregnant women and explore the correlation between different anti-HCV immunoassays, we investigated 2 independent groups in Taipei: 1,687 pregnant women without screening for serum alanine aminotransferase (ALT) (group A) and 260 pregnant women with elevated ALT activity (> 45 IU/l) screened from 15,978 cases (group B). In group A, 11 women (0.65%) were found to be anti-HCV-positive by first-generation tests and 21 (1.24%) by second-generation tests, while 7 (2.69%) and 15 (5.77%) of the group B subjects were positive, respectively. The results of the second-generation assays, based either on recombinant proteins or synthetic peptides, were identical. Among the 36 second-generation anti-HCV-positive cases, 18 (86%) of the 21 cases in group A and 13 (87%) of the 15 cases in group B contained serum HCV-RNA by RT-PCR. We conclude that the prevalence of anti-HCV in pregnant Taiwanese women is 1.24%, and the prevalence is 5.77% among those with an elevated ALT level. HCV-RNA is present in 86% of the cases positive for anti-HCV. The discrepancy between positive anti-HCV and negative HCV-RNA in some pregnant women suggests that anti-HCV positivity in such cases may merely represent a past HCV infection or a fluctuating viremia.