Approximately 90% of subjects with chronic hepatitis resulting from hepatitis C virus infection have hepatitis C virus RNA in serum. However, the prevalence of hepatitis C virus RNA in serum from subjects with hepatitis C virus antibody associated with persistent normal liver biochemical values is unclear. Do these subjects have resolved or continuing infection with hepatitis C virus? The aim of this study was to examine whether subjects with hepatitis C virus antibody but normal ALT levels had evidence of ongoing infection. Our study population was divided into four groups. Groups 1,2 and 3 comprised hepatitis C virus antibody-positive volunteer blood donors. Group 1 was made up of subjects found to be hepatitis C virus antibody-positive on enzyme-linked immunosorbent assay with persistent abnormal ALT levels (59 donors: 53 positive on recombinant immunoblot assay and 6 indeterminate). Group 2 members were hepatitis C virus antibody positive, with persistent normal ALT levels (50 donors: 39 positive on recombinant immunoblot assay and 11 indeterminate). Group 3 members were hepatitis C virus seropositive but negative on second-generation recombinant immunoblot assay (n = 48). Twenty patients (not blood donors) with chronic liver disease who were anti-hepatitis C virus seronegative were used as controls (group 4). Serum samples from all four groups were assayed for hepatitis C virus RNA on reverse transcription and a 40-cycle polymerase chain reaction with a combination of primers from the highly conserved 5'-noncoding and less-conserved third and fourth nonstructural regions. All assays were confirmed on hybridization with an internal probe. In group 1,55 of 59 subjects (93.2%) had hepatitis C virus RNA in serum. In group 2, 21 of 50 subjects (42%) had hepatitis C virus RNA in serum. Twenty-one of the 39 recombinant immunoblot assaypositive subjects (53.9%) had hepatitis C virus RNA, compared with none of the 11 recombinant immunoblot assay-indeterminant subjects (p = 0.001 on f assay). In recombinant immunoblot assay-positive subjects with normal ALT readings less than 1 mo