NAD(P)+-stimulated Ca2+ efflux from mitochondria in a high-sucrose medium is irreversible and is accompanied by large-amplitude mitochondrial swelling and membrane damage. If sucrose is partially replaced by polyethylene glycol (Mr approximately equal to 1000) as osmolar supporting medium, Ca2+ efflux is still stimulated by NAD(P)+ but mitochondrial swelling is eliminated. Other experiments in a high-sucrose medium showed that the lag phase between NAD(P)H oxidation and the beginning of net Ca2+ efflux decreases with increasing temperature. At 37 degrees C Ca2+ efflux precedes mitochondrial swelling, even in a high-sucrose medium, showing that the mitochondrial damage, as reflected by large-amplitude swelling, is not obligatory for Ca2+ efflux induced by the oxidized state of mitochondrial NAD(P)+. If a high-sucrose medium is supplemented with 20 mM potassium acetate, longer periods of Ca2+ release can be observed before the appearance of swelling. Under these experimental conditions the release of Ca2+ can be completely reversed if the rereduction of NAD(P)+ is brought about by the addition of the reductants beta-hydroxybutyrate and isocitrate.