For induction of the mitochondrial permeability transition (PT) by Ca2+, the addition of a respiratory substrate such as succinate is required. However, earlier studies indicated the possible induction of the mitochondrial PT by Ca2+ in the absence of a respiratory substrate (Hunter, D.R., and Haworth, R.A. (1979) Arch. Biochem. Biophys. 195, 453–459). In the present study, we obtained clear evidence showing that the mitochondrial PT could be induced by Ca2+ even in the absence of respiratory substrate. We next examined the protein release from mitochondria that accompanied the induction of PT in the absence of a respiratory substrate. Interestingly, distinct from the ordinary mitochondrial PT induced by Ca2+ in the presence of a respiratory substrate, which is associated with the release of mitochondrial cytochome c and adenylate kinase, the mitochondrial PT occurring in the absence of a respiratory substrate was associated with release of mitochondrial adenylate kinase but not with that of mitochondrial cytochrome c. This experimental system should be quite useful for understanding the mechanisms of protein release from mitochondria.