An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of 1 subtype of mitochondrial antibodies (AMA) specific for chronic cholestatic inflammatory liver disease. AMA were detected by ELISA in 16 of 16 patients with primary biliary cirrhosis (PBC) and in 2 of 31 patients with chronic active hepatitis. These 18 positive sera were positive for AMA by indirect immunoflourescence (IF) and by radioimmunoassay (RIA). No AMA were detected by ELISA in 2 patients with the pseudolupus erythematosus syndrome, who were positive for AMA by IF, 2 patients with secondary syphilis, positive for cardiolipin antibodies, 1 patient with systemic lupus erythematosus, positive for AMA by IF, 58 further patients with various hepatic and non-hepatic diseases and 10 healthy blood donors. The titers obtained by ELISA, ranging from 1:20 to 1:62,500, correlated well with those obtained by IF and RIA. The ELISA detected an AMA directed against one determinant of a mitochondrial antigen bearing the characteristics of the so-called PBC antigen (M2 antigen). The ELISA described is a sensitive and specific test for the detection of AMA directed against the PBC (M2) antigen and may be used not only as a standard method assaying this clinically important subtype of AMA but also as a tool for further purification and characterization of the PBC (M2) antigen.