Reoxygenation-induced release of mitochondrial aspartate aminotransferase (mAST) into the cytosol was studied using perfused rat liver. As the absolute activity of mAST in the perfusate did not indicate the degree of mitochondrial enzyme release, the following 3 methods were applied: measurement of the mAST to total AST ratio in the efferent perfusate, the digitonin infusion method, and measurement of mAST activity in the cytosolic compartment isolated from perfused livers. The results by all 3 methods were consistent and showed that mitochondrial injury occurs on reoxygenation. The mitochondrial Ca2+ content was proportional to the extent of mAST release during reoxygenation, indicating involvement of Ca2+ in the enzyme release. CsA, a potent inhibitor of Ca(2+)-induced increase in permeability of the mitochondrial membrane, completely prevented mAST release on reoxygenation. We conclude that during reoxygenation of hypoxic liver, mAST leaks into the cytosol in a Ca(2+)-dependent, CsA-sensitive manner.