We designed a rapid, homogeneous assay for human aspartate aminotransferase (AST) isoenzymes, by a selective proteolysis of soluble AST (s-AST), using chymotrypsin and protease 401. The linearity of mitochondrial (m-AST) was elongated up to 4000 U/l. m-AST values from the human liver, and determined by a homogeneous assay using protease 401 or chymotrypsin, were relative to those obtained using an immunoprecipitation method. In perioperative patients or those with an acute myocardial infarction, the peaks of s-AST and m-AST values were noted 13 h and at 57 h after ictus, respectively, whereas the peak of ratio between was seen 6 h after ictus. In the case of Budd-Chiari syndrome, the maximum levels of the two AST activities were evident 14 days after hospitalization and the peak of ratio between them was seen after 7 days. We propose that this homogeneous assay can serve as a diagnostic tool for early phase detection of myocardial infarction and of Budd-Chiari syndrome.