We have developed an enzyme linked immunosorbant assay (ELISA) for antimitochondrial antibody. Polyvinyl microtiter plate wells are coated with partially purified rat kidney mitochondria, and excess protein binding sites are blocked with bovine serum albumin. Human serum, diluted 1:1,000, is incubated for 1 hr. Then β-galactosidase-goat-anti-human IgG (H+L) is added followed by the substrate, p-nitrophenyl-β-D-galactopyranoside. The plates are then read at 404 nM in a microelisa autoreader. A positive result was defined as optical density ± 0.100, more than 5 standard deviations above the mean of 36 normal individuals. With this techique, 56 of 60 patients with primary biliary cirrhosis were positive for antimitochondrial antibody (93%), mean O.D., 0.456 ± 0.031 S.E. Seventeen of 17 patients with extrahepatic bile duct obstruction and 14 or 14 patients with alcoholic cirrhosis were negative. Only 1 of 29 patients with chronic active liver disease was positive (4%). Antinuclear antibody and antimicrosomal antibody do not bind in this assay, and activity is absorbed from sera by preincubation with suspensions of rat kidney mitochondria. The ELISA is approximately 20 times more sensitive than a quantitive microtiter complement fixation technique and more convenient than radioimmunoassay. It is rapid, quantitative and uses stable reagents. In contrast to immunofluorescence techniques, it is not affected by observer interpretation.