To compare different hepatitis C virus (HCV) immunoassays and HCV-RNA in pregnant women, we investigated two independent groups: 1,687 cases without screening for serum alanine aminotransferase (ALT) (group A) and 333 cases with elevated ALT (>45 IU/1) (group B), after screening 21,459 pregnant women. In group A, 11 (0.65%) and 21 (1.24%) were anti-HCV-positive by first- and second-generation tests, respectively, while in group B 8 (2.40%) and 19 (5.71%) were positive, respectively. The results revealed by second-generation assays based on either recombinant protein or synthetic peptides were identical, as were the anti-HCV titers in group B. Among 40 second-generation anti-HCV-positive cases, 18 (86%) of 21 in group A and 17 (89%) of the 19 in group B contained serum HCV-RNA by RT-PCR. Thus the prevalence of anti-HCV in Taiwanese pregnant women is 1.24% versus 5.71% in those with elevated ALT level.