The mechanism of external NADH oxidation in rat liver mitochondria is not clear yet, but it evidently includes the intermembrane electron transport (IMET). We showed that Mg 2+ significantly stimulated IMET insensitive to rotenone–antimycin A–myxothiazol in mitochondria with the hypotonically damaged outer membrane, even in the absence of exogenous cytochrome c . This effect was more specific than simply ionic strength increase. Direct evidence for cytochrome c desorption from mitochondrial membranes under 4 mM Mg 2+ influence was obtained to explain the stimulatory effect of Mg 2+ on external NADH oxidation that was coupled to the inner membrane potential generation. Obtained data suggest that cytochrome b 5 is normally oriented towards the cytosol in the outer membrane, and can be accessible for endogenous cytochrome c reduction only through the outer membrane rupture or permeabilization, to activate external NADH oxidation.