The ATP-regulated potassium channel is present in the inner membrane of heart mitochondria. In this study, the activity of a single channel was measured after reconstituting the myocardium inner mitochondrial membrane into a planar lipid bilayer. We provide direct evidence of vectorial pH regulation of mitoK(ATP) channels. When the matrix side was alkalized, this changed the channel conductance, the open probability, and the mean open and closed dwell time distributions. The conductance of the mitoK(ATP) channel increased from about 110 +/- 8 to 145 +/- 5 pS upon changing the pH from 7.2 to 8.2. This effect was reversed by reverting the pH to the neutral value. The mitoK(ATP) channel activity was not altered by alkalization of the cytosolic side of the planar lipid bilayer. We also observed that acidification from pH 7.2 to 6.2, in either the matrix or cytosolic compartments, decreased the open probability of the channel. This effect was reversed by perfusion with a pH 7.2 medium. Additionally, our results suggest that the mitoK(ATP) channel is regulated by multiple phosphorylation events. The channel activity was inhibited by an ATP/Mg(2+) complex, but not by ATP alone, nor by a non-hydrolysable ATP analog, e.g. AMP-PNP/Mg(2+). The mitoK(ATP) channel "run-down" was reversed by incubating with the ATP/Mg(2+) complex on both sides of the planar lipid bilayer. We conclude that both pH and ATP play an important regulatory role for the cardiac mitoK(ATP) channel with respect to the phenomenon of ischemia-reperfusion.