ABSTRACT— The expression of large (pre-S1), middle (pre-S2), major S (S) polypeptides of the envelope (HBs) and X peptides of hepatitis B virus (HBV) was investigated in 37 liver specimens with chronic hepatitis B by indirect immunoperoxidase staining. Primary antisera utilized were polyclonal ones against HBs (poly-HBs), core (HBc) and X and monoclonal ones against pre-Sl, pre-S2 and S with (particle-S) or without (peptide-S) conformational structure. The localization of HBs proteins in hepatocytes was classified into three types: diffuse, membranous and inclusion. The peptide-S and pre-S2 were expressed at nearly the same frequency as poly-HBs in all types, whereas particle-S was found less frequently (18/29 cases) in the inclusion type, and pre-S1 was recognized relatively rarely (9/33 cases) in the membranous type. As for staining intensity, peptide-S and pre-S2 were almost identical to poly-HBs which stained the most strongly among all three staining types. Particle-S was similar to poly-HBs in the membranous type, but was weak in the inclusion type in the majority. While pre-S1 was stained in a similar intensity to poly-HBs in the diffuse and inclusion types, it was weak or negative in the membranous type. Thus, envelope particles indicated by particle-S staining appeared to be located most frequently in the membranous type, but their assembly might be suppressed in the inclusion type where pre-S1 was well expressed. The X peptide was more frequently detected in the liver with serum HBe antigen and/or HBV DNA. The X peptide was stained exclusively in the cytoplasm of hepatocytes and was correlated with the cytoplasmic HBc antigen. The X peptide was not observed differently between cases with and those without cirrhosis. This suggests that the expression of X peptide tends to occur with virus replication but not with disease progression.