The purpose of this study was to compare the serological analysis for hepatitis C infection using the recombinant immunoblot assay (RIBA) to the direct in situ localization of the hepatitis C viral genome using the reverse transcriptase (RT) in situ PCR technique. Concurrent sera and liver biopsies from 42 patients with clinical and histologic evidence of chronic liver disease were studied. Antibodies against hepatitis C specific antigens were demonstrated by RIBA in the sera of 39/42 (92%), and PCR amplified viral cDNA was detected in the biopsies of 21 (54%) of the 39 seropositive patients. The detection rate using standard in situ hybridization for the tissues known to be viral positive with RT in situ PCR was 9/21 (42%). It is concluded that approximately one-half of patients with chronic hepatitis and serologic evidence of hepatitis C infection will not have virus detectable in their liver biopsy even with a highly sensitive PCR-based technique.