To comprehensively study autoantibodies in patients with chronic hepatitis (CH), especially those with hepatitis C virus (HCV) infection, proteins extracted from HepG2 cells were separated by two-dimensional electrophoresis. Spots reacting with sera from 15 patients with CH-C were detected by Western blotting. Proteins extracted from the spots were subjected to mass spectroscopy for identification by mass fingerprinting. Antigenicity of the proteins identified was confirmed by Western blotting and enzyme-linked immunoabsorbent assay. The localization of the autoantigens so detected was investigated by immunohistochemistry. Among 20 protein spots detected, four were identified as actin, heat shock protein (HSP) 70, HSP60, and a novel protein (hepalaminin). Hepalaminin consists of two domains of laminin beta-2 and a specific domain. Autoantibodies against the specific domain were detected in 60.8% of patients with CH-C, 37.7% of those with CH-B, 42.3% of those with autoimmune CH, 28.6% of nonalcoholic steatohepatitis, 10.0% of asymptomatic HCV carriers, but in no healthy volunteers. Antihepalaminin positivity in CH-C and CH-B was related to histologic grading. Immunohistochemical staining demonstrated that hepalaminin is present in the cytoplasm of hepatocytes and cholangiocytes but not of fibroblasts or the vascular epithelium. Hepalaminin is a novel protein expressed in hepatocytes and cholangiocytes. Autoimmunity to this protein may exacerbate inflammation in chronic viral hepatitis.