The mitochondrial ATP-sensitive K+ channel (mitoK(ATP)) has been assigned multiple roles in cell physiology and in cardioprotection. Each of these roles must arise from basic consequences of mitoK(ATP) opening that should be observable at the level of the mitochondrion. MitoK(ATP) opening has been proposed to have three direct effects on mitochondrial physiology: an increase in steady-state matrix volume, respiratory stimulation (uncoupling), and matrix alkalinization. Here, we examine the evidence for these hypotheses through experiments on isolated rat heart mitochondria. Using perturbation techniques, we show that matrix volume is the consequence of a steady-state balance between K+ influx, caused either by mitoK(ATP) opening or valinomycin, and K+ efflux caused by the mitochondrial K+/H+ antiporter. We show that increasing K+ influx with valinomycin uncouples respiration like a classical uncoupler with the important difference that uncoupling via K+ cycling soon causes rupture of the outer mitochondrial membrane and release of cytochrome c. By loading the potassium binding fluorescent indicator into the matrix, we show directly that K+ influx is increased by diazoxide and inhibited by ATP and 5-HD. By loading the fluorescent probe BCECF into the matrix, we show directly that increasing K+ influx with either valinomycin or diazoxide causes matrix alkalinization. Finally, by comparing the effects of mitoK(ATP) openers and blockers with those of valinomycin, we show that four independent assays of mitoK(ATP) activity yield quantitatively identical results for mitoK(ATP)-mediated K+ transport. These results provide decisive support for the hypothesis that mitochondria contain an ATP-sensitive K+ channel and establish the physiological consequences of mitoK(ATP) opening for mitochondria.