Testing three anti-M2/anti-M4-positive and three anti-M2 positive/anti-M4-negative primary biliary cirrhosis (PBC) marker sera against different mitochondrial enzymes by ELISA it could be shown that only the anti-M4-positive sera reacted with pyruvate dehydrogenase (M2) and sulphite oxidase (SO), an enzyme of the mitochondrial inter-membrane space in parallel. Absorption of these sera with SO abolished completely the anti-M4 antibodies but had no effect on the anti-M2 activity. The specificity of this reaction was also documented by examining 30 anti-M2/anti-M4-positive sera showing that 28 of them were positive with SO. Among ten anti-M2/anti-M8-positive but anti-M4-negative PBC sera, four became positive when tested against SO, indicating a higher sensitivity of SO for the demonstration of anti-M4. Retesting sera from 76 PBC patients with defined anti-mitochondrial antibody (AMA) profiles who had been followed for up to 18 years against SO by ELISA and complement fixation test (CFT), none of 32 patients with profile A B (positive for anti-M2 and/or anti-M9 by ELISA; benign course) but 33 of 44 patients with profile C/D (anti-M2/anti-M4 and or anti-M8 positive by CFT and or ELISA; progressive course) were positive. These data indicate that sulphite oxidase can be used in the ELISA for the detection of anti-M4 antibodies which may be of prognostic relevance.