A dot-immunobinding assay was established for the detection of antimitochondrial antibodies. Nitrocellulose strips were coated with sonicated rat liver mitochondria and incubated in the presence of human sera. The resulting immune complexes were visualized with an enzyme-linked second antibody. Antimitochondrial antibodies were found in the sera of 96% of patients with primary biliary cirrhosis, 17% of patients with autoimmune hepatitis and 4% of patients with progressive systemic sclerosis. Sera of patients with other liver diseases or with systemic lupus erythematosus, specimens positive for M1 and M3 mitochondrial antibodies and samples from normal controls were all negative. Antinuclear and other cytoplasmic antibodies were not detected in this assay. The dot-immunobinding assay for antimitochondrial antibodies is rapid and sensitive, and obviates the need for expensive equipment.