Anti-mitochondrial antibodies (AMA) were detected by indirect immunofluorescence in the sera of 16 out of 17 (94%) patients with primary biliary cirrhosis (PBC). Immunoblotting experiments with mitochondrial polypeptides from the porcine liver as antigens revealed that three antigens were recognized by the sera from AMA-positive patients. These were a 70-kD protein recognized by nine out of 16 AMA-positive sera, a 50-kD protein recognized by 13 out of 16 AMA-positive sera and a 39-kD protein recognized by four out of 16 AMA-positive sera. The reactivity of these polypeptides was destroyed by brief exposure to trypsin. None of these antigens were recognized by any of the 30 control sera. These results show that the 70-kD, 50-kD and 39-kD proteins are the major mitochondrial autoantigens recognized by sera from patients with PBC. In addition, of 30 sera samples from patients with diffuse scleroderma, 13 reacted to the 70-kD and/or 50-kD antigens. Anti-centromere antibodies (ACA) were also detected in the sera of five of the 17 (29%) patients with PBC. The high prevalence of ACA in patients with PBC and the presence of anti-70- and 50-kD antibodies in patients with diffuse scleroderma provide evidence of an association between these two disorders.