Hepatitis C virus (HCV) antigen was detected immunohistochemically using fluorescein isothiocyanate-labeled immunoglobulin G fractions from chimpanzee and human sera strongly reactive with recombinant hepatitis C virus structural and non-structural proteins. The antigen was localized in the cytoplasm of hepatocytes in all 9 chimpanzees with acute hepatitis C, in 5 of 10 chimpanzees with chronic HCV infection, and in 11 of 12 patients with chronic hepatitis C. The specificity of the hepatocellular HCV and FITC-labeled probes for HCV was ascertained by blocking studies with paired serum samples obtained from 8 infected and uninfected chimpanzees or from 14 patients during the acute and chronic phases of HCV infection. Absorption experiments on FITC-labeled probes with selected host proteins (normal liver homogenate, plasma proteins, red blood cells) did not indicate cross reactivity of the probes with these antigens. Direct immunomorphologic evidence for the HCV specificity of hepatocellular HCV antigen deposits and the FITC-labeled polyclonal anti-HCVAg probe was established in absorption experiments using recombinant HCV nonstructural proteins. The putative HCV NS3 protein was the most prominent component of hepatocellular HCV antigen.