The pharmacological phenotype of ATP-sensitive potassium (K(ATP)) channels is defined by their tissue-specific regulatory subunit, the sulfonylurea receptor (SUR), which associates with the pore-forming channel core, Kir6.2. The potassium channel opener diazoxide has hyperglycemic and hypotensive properties that stem from its ability to open K(ATP) channels in pancreas and smooth muscle. Diazoxide is believed not to have any significant action on cardiac sarcolemmal K(ATP) channels. Yet, diazoxide can be cardioprotective in ischemia and has been found to bind to the presumed cardiac sarcolemmal K(ATP) channel-regulatory subunit, SUR2A. Here, in excised patches, diazoxide (300 microM) activated pancreatic SUR1/Kir6.2 currents and had little effect on native or recombinant cardiac SUR2A/Kir6.2 currents. However, in the presence of cytoplasmic ADP (100 microM), SUR2A/Kir6.2 channels became as sensitive to diazoxide as SUR1/Kir6. 2 channels. This effect involved specific interactions between MgADP and SUR, as it required Mg(2+), but not ATP, and was abolished by point mutations in the second nucleotide-binding domain of SUR, which impaired channel activation by MgADP. At the whole-cell level, in cardiomyocytes treated with oligomycin to block mitochondrial function, diazoxide could also activate K(ATP) currents only after cytosolic ADP had been raised by a creatine kinase inhibitor. Thus, ADP serves as a cofactor to define the responsiveness of cardiac K(ATP) channels toward diazoxide. The present demonstration of a pharmacological plasticity of K(ATP) channels identifies a mechanism for the control of channel activity in cardiac cells depending on the cellular ADP levels, which are elevated under ischemia.