The effects of hydrogen peroxide (H2O2) on the activity of adenosine 5'-triphosphate-sensitive potassium (K+ ATP) channels in ventricular cells isolated from guinea-pig hearts were investigated in inside-out membrane patches using the patch-clamp technique. H2O2 at concentrations of 5 mM but not 1 mM increased K+ ATP channel activity in the presence of 0.3 mM ATP. The presence of 10 microM ADP together with 0.3 mM ATP led to activation of the K+ ATP channel by 1 mM H2O2. This potentiation of the H2O2-induced activation of the K+ ATP channel by ADP depended on the presence of Mg-ATP. Channel activation was due to an increase in the open-state probability and was not associated with a change in the single-channel conductance or the mean open and closed times during burst-like openings. The relationship between channel activity and ATP concentration could be fitted by the Hill equation with a Hill coefficient of 2 and the half maximal inhibition at 85 microM ATP in the presence of 10 microM ADP. The curve was shifted to higher ATP concentrations in a non-parallel manner by 1 mM H2O2. Analysis of open-state probability for membrane patches containing several channels indicated that H2O2 activated the individual channels to a differing extent. It is concluded that H2O2 activates the K+ ATP channel directly by decreasing the sensitivity to ATP. This effect was potentiated by the presence of ADP.