1. The ATP-regulated potassium channel (K+ATP) was investigated with respect to modulation by intracellular pH (pHi) by using the inside-out membrane patch clamp technique in ventricular cells isolated from the heart of the guinea-pig. Channels which had been closed by internal ATP (0.3-3 mM) were dose-dependently activated by decreasing the pHi over the range of pH 7.6-6.0. However, the channel was conversely inhibited when the pHi was further decreased below 6.0. Inwardly rectifying K+ channels were also decreased in activity when pHi fell from 7.2 to 6.0. 2. The channel activation was also observed with constant concentration of free Ca2+ (1 nM) and Mg2+ (1 mM) in the bathing solution, suggesting that a change in divalent cation concentration is not involved in channel modulation by pHi. 3. When the dose-response relations of the channel activity for ATP concentrations at different pHi were examined, the channel activity obtained at 1 microM ATP was increased by decreasing pH from 7.2 to 6.4. The half-maximal inhibition for ATP concentration at pH 7.2 and 6.4 was 20 and 40 microM, respectively, and the Hill coefficient was 2.5 in both curves. 4. In the absence of ATP, internal H+ was able to reactivate run-down channels but it had less effect on the channel as long as the activity was maintained at a higher level. The increase in the channel activity by H+ was facilitated with a proceeding of the run-down. However, after the channel was completely inactivated by a long exposure of the membrane patch to ATP-free solution, a reduction of pH could not activate the channel. 5. The decrease of pH from 7.2 to 6.4 reduced single channel conductance from 89.0 to 77.7 pS in the absence of Mg2+, whereas it reduced the conductance only at the negative membrane potentials in the presence of 2 mM Mg2+. 6. Mean open and closed times within the burst-like openings of the channel remained unaffected during the change in pHi. 7. We conclude that the cardiac K+ATP channel is modulated by a change in the intracellular pH. The channel modulation consisted of the increase in the channel activity and a decrease in the permeability. The former effect was due to the decrease in the sensitivity of the channel to ATP and the reactivation of the channel which is during the process of run-down in activity.