An antigen (HEVAg) associated with enterically transmitted non-A, non-B (ET-NANB) hepatitis virus has been identified in hepatocytes of cynomolgus macaques (Macaca fascicularis) infected with ET-NANB hepatitis by using an immunofluorescent probe prepared from serum previously shown by immune electron microscopy to react with ET-NANB viruslike particles. HEVAg was observed in the cytoplasm of hepatocytes in 27 of 29 infected macaques in five passages of the diseases but not in uninoculated macaques or chimpanzees with various types of experimentally induced viral hepatitis. Specificity of the assay was confirmed by blocking tests using preinoculation, acute, and convalescent sera from ET-NANB hepatitis patients and from experimentally infected macaques. Concordant results of fluorescent antibody blocking and immune electron microscopy assays suggest that hepatocellular HEVAg is associated with 32- to 34-nm viruslike particles etiologically linked to disease. Antibodies to HEVAg were identified, and the titers were determined by using a fluorescent antibody blocking assay on liver tissue sections from macaques infected with ET-NANB hepatitis.