The mitochondrial KATP channel (mitoKATP) is highly sensitive to ATP, which inhibits K+ flux with K1/2 values of 20-40 microM. This raises the question, how can mitoKATP be opened in the presence of physiological concentrations of ATP? We measured K+ flux in liposomes reconstituted with purified mitoKATP and found that guanine nucleotides are potent activators of this channel. ATP-inhibited K+ flux was completely reactivated by both GTP (K1/2 = 7 microM) and GDP (K1/2 = 140 microM). These ligands had no effect in the absence of ATP. The K1/2 for ATP inhibition exhibited quadratic dependence on [GTP] and [GDP], consistent with two binding sites for guanine nucleotides. We also found that palmitoyl-CoA and oleoyl-CoA inhibited K+ flux through reconstituted mitoKATP with K1/2 values of 260 nM and 80 nM, respectively. This inhibition was reversed by GTP (K1/2 = 232 microM) as well as by the K+ channel openers cromakalim (20 microM) and diazoxide (10 microM). Inhibition of mitoKATP by long-chain acyl-CoA esters, like that of ATP, exhibited an absolute requirement for Mg2+ ions. We propose that the open-closed state of the mitochondrial KATP channel is determined by the relative cytosolic concentrations of GTP and long-chain acyl-CoA esters.