A new antimitochondrial antibody (AMA) against an outer membrane associated antigen on liver mitochondria was detected by ELISA in sera from patients with primary biliary cirrhosis (PBC). This antibody was named anti-M9. There is evidence that it is a partial organ-specific antibody as shown by absorption studies using submitochondrial particles prepared from heart, liver and kidney. A purified M9-fraction was prepared by subjecting a 100,000 g supernatant from rat liver mitochondria to ion exchange chromatography. This fraction was devoid of the previously described M1-M8 antigens except for M4. Trypsin treatment of the fraction enabled a distinction to be made between M4 which was protease resistant, and M9 which was trypsin sensitive. Applying this M9-fraction in Western blotting anti-M9 positive sera recognized two proteins at a molecular weight of 98 kD and 59 kD. Anti-M9 antibodies were detected in 37% of 156 anti-M2 positive as well as in 82% of 22 anti-M2 negative patients with histologically proven PBC. It is concluded that anti-M9 is a new AMA type in PBC which may be helpful especially for the early diagnosis of PBC in patients who are still anti-M2 negative. As one of the earliest immunological signs in PBC further characterization of M9 could provide new insights into the etiopathogenesis of the disease.