A radioimmunoassay (RIA) was established for the detection of antimitochondrial autoantibodies (AMAs) in patient sera. AMAs were detected by RIA in 12 of 14 patients with primary biliary cirrhosis and in 3 of 29 patients with chronic active hepatitis. AMAs were detected by indirect immunofluorescence in all sera positive by RIA. In addition, two patients with primary biliary cirrhosis and one patient with chronic active hepatitis were AMA positive when tested by indirect immunofluorescence, but negative when tested by RIA. AMAs were not detected by RIA or indirect immunofluorescence in 121 further patients with various hepatic and nonhepatic diseases, including healthy controls. The RIA detects an AMA directed against one determinant of a mitochondrial antigen which bears the characteristics of the previously described primary biliary cirrhosis antigen (M2 antigen). The RIA described is a sensitive and specific test for the detection of one mitochondrial autoantibody associated with chronic cholestatic liver diseases displaying the morphology of primary biliary cirrhosis in chronic active hepatitis or liver biopsy specimens. Antibodies against other mitochondrial antigens do not react in this assay system.