The ATP-sensitive K+ (K(ATP)) channels are regulated by intracellular H+ in addition to ATP, ADP, and phospholipids. Here we show evidence for the interaction of H+ with ATP in regulating a cloned K(ATP) channel, i.e. Kir6.2 expressed with and without the SUR1 subunit. Channel sensitivity to ATP decreases at acidic pH, while the pH sensitivity also drops in the presence of ATP. These effects are more evident in the presence of the SUR1 subunit. In the Kir6.2 + SUR1, the pH sensitivity is reduced by about 0.4 pH units with 100 microM ATP and 0.6 pH units with 1 mM ATP, while a decrease in pH from 7.4 to 6.8 lowers the ATP sensitivity by about fourfold. The Kir6.2 + SUR1 currents are strongly activated at pH 5.9-6.5 even in the presence of 1 mM ATP. The modulations appear to take place at His175 and Lys185 that are involved in proton and ATP sensing, respectively. Mutation of His175 completely eliminates the pH effect on the ATP sensitivity. Similarly, the K185E mutant-channel loses the ATP-dependent modulation of the pH sensitivity. Thus, allosteric modulations of the cloned K(ATP) channel by ATP and H+ are demonstrated. Such a regulation allows protons to activate directly the K(ATP) channels and release channel inhibition by intracellular ATP; the pH effect is further enhanced with a decrease in ATP concentration as seen in several pathophysiological conditions.