P1P6-di(adenosine-5')hexaphosphate (Ap6A) has been recently demonstrated in mammalian cells, yet its function remains unknown. Using single-channel current recordings, we studied the effect of Ap6A on guinea-pig cardiac ATP-sensitive K+ (K(ATP) channels. When applied to the intracellular side of excised membrane patches, Ap6A produced a concentration-dependent inhibition (half-maximal inhibitory concentration: 14 microM; Hill coefficient: 1.1) of K(ATP) channel activity. We conclude that Ap6A is a potent inhibitory ligand of K(ATP) channels.