In this study, the effects of polyamines and analogous compounds on mitochondrial permeability transition were characterized to distinguish between these effects and those on mitochondrial Ca 2+ uptake, which are described in an accompanying report (Rustenbeck et al., Biochem Pharmacol 8: 977–985, 1998). When a transitional Ca 2+ release from Ca 2+-loaded mitochondria was induced by an acute increase in Ca 2+ concentration in a cytosol-adapted incubation medium (Ca 2+ pulse), this process was inhibited, but not abolished by spermine in the concentration range of 0.4 to 20 mM. The aminoglucoside, gentamicin, and the basic polypeptide, poly- l-lysine, which like spermine are able to enhance mitochondrial Ca 2+ accumulation (preceding paper), had no or only a minimal inhibitory effect, while the aliphatic polyamine, bis(hexamethylene)triamine, which is unable to enhance mitochondrial Ca 2+ accumulation, achieved a complete inhibition at 4 mM. The conclusion that the Ca 2+ efflux was due to opening of the permeability transition pore was supported by measurements of mitochondrial membrane potential, ATP production, and oxygen consumption. Mg 2+, a known inhibitor of mitochondrial membrane permeability transition, did not mimick the effects of spermine on mitochondrial Ca 2+ accumulation, while ADP, the main endogenous inhibitor, showed both effects. However, a combination of spermine and ADP was significantly more effective than ADP alone in restoring low Ca 2+ concentrations after a Ca 2+ pulse. Two different groups of spermine binding sites were found at intact liver mitochondria, characterized by dissociation constants of 0.5 or 4.7 mM and maximal binding capacities of 4.6 or 19.7 nmol/mg of protein, respectively. In contrast to aminoglucosides, the aliphatic polyamine bis(hexamethylene)triamine did not displace spermine from mitochondrial binding sites. The total intracellular concentration of spermine in hepatocytes was measured to be ca. 450 μM and the free cytoplasmic concentration was estimated to be in the range of 10–100 μM. In conclusion, the enhancement of mitochondrial Ca 2+ uptake by spermine is not an epiphenomenon of the inhibition of permeability transition. The physiological role of spermine appears to be that of an enhancer of mitochondrial Ca 2+ accumulation rather than an inhibitor of permeability transition.