Inwardly rectifying K(+) (K(ir)) channels in the apical membrane of the retinal pigment epithelium (RPE) contribute to extracellular K(+) homeostasis in the distal retina by mediating K(+) secretion. Multiple lines of evidence suggest that these channels are composed of Kir7.1. Previously, we showed that native K(ir) channels in bovine RPE are modulated by changes in intracellular pH in the physiological range. In the present study, we used the Xenopus laevis oocyte expression system to investigate the pH dependence of cloned human Kir7.1 channels and several point mutants involving histidine residues in the NH(2) and COOH termini. Kir7.1 channels were inhibited by strong extracellular acidification and modulated by intracellular pH in a biphasic manner, with maximal activity at about intracellular pH (pH(i)) 7.0 and inhibition by acidification or alkalinization. Replacement of histidine 26 (H26) in the NH(2) terminus with alanine eliminated the requirement of protons for channel activity and increased sensitivity to proton-induced inhibition, resulting in maximal channel activity at alkaline pH(i) and smaller whole cell currents at resting pH(i) compared with wild-type Kir7.1. When H26 was replaced with arginine, the pH(i) sensitivity profile was similar to that of the H26A mutant but with the pK(a) shifted to a more acidic value, giving rise to whole cell current amplitude at resting pH(i) that was comparable to that of wild-type Kir7.1. These results indicate that Kir7.1 channels are modulated by intracellular protons by diverse mechanisms and suggest that H26 is important for channel activation at physiological pH(i) and that it influences an unidentified proton-induced inhibitory mechanism.