The mechanisms of leakage of intracellular enzymes, and especially the cytosolic and mitochondrial isozymes of aspartate aminotransferase (AST), in ischemic rat liver were studied. On recirculation of ischemic liver, cytosolic AST (cAST) promptly appeared in the blood. Release of cytosolic enzymes, including cAST and lactic dehydrogenase, resulted from disruption of blebs that protruded from parenchymal cells into the sinusoidal space. When these blebs were formed in ischemic liver, mitochondria still remained in core regions of the injured cells and were not found in the blebs. Consistent with this fact, mitochondrial AST (mAST) did not leak into the circulation from ischemic liver until most of the cAST had leaked out. This delayed leakage of mitochondrial enzymes was also consistent with the fact that the mitochondrial membranes maintained a diffusion barrier against matrix enzymes even after anoxia for 2 h, when their oxidative phosphorylation capacity had been lost. These results indicate that mitochondrial enzymes are liberated into the blood only after appreciable disintegration of the cells, probably necrosis, and that the cumulative activity of mAST in the blood should reflect the extent of necrosis in ischemic organs better than that of cAST.