K+ currents were recorded from ATP-sensitive channels in inside-out membrane patches excised from isolated rat ventricular myocytes. ATP-sensitive K+ channel inhibition could be evoked by ATP in the absence of magnesium where most ATP would be present as the free acid ATP4-. Channel inhibition was enhanced when the same total concentration of ATP was applied in the presence of magnesium, where most ATP would be bound as ATP.Mg. Dose-response relationships for ATP-sensitive K+ channel inhibition evoked by ATP had a Hill coefficient of 2 and Ki of 17 and 30 microM for ATP in the presence and absence of magnesium respectively. This was the obverse of the expected results if ATP4- were to be the sole form of ATP to effect channel closure. ATP-sensitive K+ channel inhibition evoked by ATP gamma S, AMP-PNP and AMP-PCP was also enhanced in the presence of magnesium. It is concluded that the ATP-sensitive K+ channel of rat ventricular myocytes binds and is closed by both the free-acid and divalent-cation-bound forms of ATP.