The rubidium efflux from hypothermic rat hearts perfused by the Langendorff method at 20°C was studied. At this temperature ⁸⁷Rb-NMR efflux experiments showed the existence of two ⁸⁷Rb pools: cytoplasmic and mitochondrial. Rat heart mitochondria showed a very slow exchange of mitochondrial Rb⁺ for cytoplasmic K⁺. After washout of cytosolic Rb⁺, mitochondria kept a stable Rb⁺ level for >30 min. Rb⁺ efflux from mitochondria was stimulated with 0.1 mM 2,4-dinitrophenol (DNP), by sarcolemmal permeabilization and concomitant cellular energy depletion by saponin (0.01 mg/ml for 4 min) in the presence of a perfusate mimicking intracellular conditions, or by ATP-sensitive K (K_ATP) channel openers. DNP, a mitochondrial uncoupler, caused the onset of mitochondrial Rb⁺ exchange; however, the washout was not complete (80 vs. 56% in control). Energy deprivation by saponin, which permeabilizes the sarcolemma, resulted in a rapid and complete Rb⁺ efflux. The mitochondrial Rb⁺ efflux rate constant (k) decreased in the presence of glibenclamide, a K_ATP channel inhibitor (5 µM; k = 0.204 ± 0.065 min¹; n = 8), or in the presence of ATP plus phosphocreatine (1.0 and 5.0 mM, respectively; k = 0.134 ± 0.021 min¹; n = 4) in the saponin experiments (saponin only; k = 0.321 ± 0.079 min¹; n = 3), indicating the inhibition of mitochondrial K_ATP channels. Thus hypothermia in combination with ⁸⁷Rb-NMR allowed the probing of the mitochondrial K⁺ pool in whole hearts without mitochondrial isolation.