The mitochondrial KATP channel (mitoKATP) is richly endowed with regulatory sites for metabolites and drugs, but the topological location of these sites is unknown. Thus, it is not known whether ATP, GTP and acyl CoA esters regulate mitoKATP from the matrix or cytosolic side of the inner membrane, nor whether they all act from the same side. The experiments reported in this paper provide an unambiguous answer to these questions. Electrophysiological experiments in bilayer membranes containing purified mitoKATP showed that current is blocked asymmetrically by ATP. K+ flux experiments using proteoliposomes containing purified mitoKATP showed that mitoKATP is unipolar with respect to regulation by Mg2+, ATP, GTP, and palmitoyl CoA and that all of these ligands react on the same pole of the protein. This demonstration was made possible by the new finding that mitoKATP is 85-90% oriented inward or outward in liposomes, depending on the presence or absence of Mg2+ in the reconstitution buffer. K+ flux experiments in respiring rat liver mitochondria showed that mitoKATP was inhibited by palmitoyl CoA and activated by GTP when these agents were added to the external medium. Given that the inner membrane is impermeant to these ligands and that mitoKATP is unipolar with respect to nucleotide regulation, it follows that the regulatory sites on mitoKATP face the cytosol.