The expression of antigens on rat hepatocytes was examined by immunoelectron microscopy using monoclonal antibody HAM 1. The antigen recognized by HAM 1 was expressed mainly on both sinusoidal and bile-canalicular faces and only sparsely on the contiguous face. All rat hepatoma cell lines examined were intensely labeled with HAM 1, but poorly labeled with HAM 3, HAM 4, and HAM 5, as revealed by flow cytofluorometry and radioimmunoassay. Expression levels of HAM 1 antigen on the AH 44 hepatoma cell line similar to the degree seen in normal liver were demonstrated by radioimmunoassay. HAM 1 antigen was also expressed weakly on lymphocytes, thymocytes, and some bone marrow cells, but not on red blood cells, and differed from the MHC class I antigen recognized by HAM 2 and OX 18. Significant amounts of cell surface antigens recognized by all the monoclonal antibodies (HAM 1-HAM 5) were confirmed by radioimmunoassay on the cell surface of primary cultured hepatocytes. These results suggest that the rat hepatoma cell lines employed are different from normal hepatocytes, that primary cultured hepatocytes are more similar to normal hepatocytes in the degree of expression of their cell surface antigens, and that HAM 1 antigen appears to be a significant antigen on both normal and transformed hepatocytes.