A mechanism of Ca²⁺ uptake, capable of sequestering significant amounts of Ca²⁺ from cytosolic Ca²⁺ pulses, has previously been identified in liver mitochondria. This mechanism, the Rapid Mode of Ca²⁺ uptake (RaM), was shown to sequester Ca²⁺ very rapidly at the beginning of each pulse in a sequence [Sparagna et al. (1995) J. Biol. Chem. 270, 27510–27515]. The existence and properties of RaM in heart mitochondria, however, are unknown and are the basis for this study. We show that RaM functions in heart mitochondria with some of the characteristics of RaM in liver, but its activation and inhibition are quite different. It is feasible that these differences represent different physiological adaptations in these two tissues. In both tissues, RaM is highly conductive at the beginning of a Ca²⁺ pulse, but is inhibited by the rising [Ca²⁺] of the pulse itself. In heart mitochondria, the time required at low [Ca²⁺] to reestablish high Ca²⁺ conductivity via RaM i.e. the ‘resetting time’ of RaM is much longer than in liver. RaM in liver mitochondria is strongly activated by spermine, activated by ATP or GTP and unaffected by ADP and AMP. In heart, RaM is activated much less strongly by spermine and unaffected by ATP or GTP. RaM in heart is strongly inhibited by AMP and has a biphasic response to ADP; it is activated at low concentrations and inhibited at high concentrations. Finally, an hypothesis consistent with the data and characteristics of liver and heart is presented to explain how RaM may function to control the rate of oxidative phosphorylation in each tissue. Under this hypothesis, RaM functions to create a brief, high free Ca²⁺ concentration inside mitochondria which may activate intramitochondrial metabolic reactions with relatively small amounts of Ca²⁺ uptake. This hypothesis is consistent with the view that intramitochondrial [Ca²⁺] may be used to control the rate of ADP phosphorylation in such a way as to minimize the probability of activating the Ca²⁺-induced mitochondrial membrane permeability transition (MPT).