A-type K+ currents serve important functions in neural and cardiac physiology. The human A-type Kv1.4 channel (hKv1.4) shows fast N-type inactivation when expressed in Xenopus laevis oocytes. We found that intracellular pH (pH(i)) regulated the macroscopic inactivation time constant (tau) and current amplitude (I(peak)), producing a 2-fold change with each pH unit change in the physiologically relevant range of 8.0 to 6.0. These effects of pH(i) were completely abolished by a large deletion in the hKv1.4 N terminus. Site-directed mutagenesis identified a histidine (H16) in the inactivation ball domain as a critical H+ titratable site mediating the pH effects on N-type inactivation between pH 7.0 and 9.0. Substituting this histidine with arginine not only accelerated the time course of macroscopic channel inactivation but also eliminated the H+ effects on hKv1.4. In addition, a glutamic acid (E2) in the ball domain constitutes another H+ titratable site that mediates the pH effects in the more acidic pH range of 5.0 to 7.0. These results suggest that N-type inactivation in hKv1.4 is regulated by pH(i) in the physiologic range through ionization of specific amino acid residues in the ball domain. Such pH(i) effects may represent an important fundamental mechanism for physiological regulation of excitable tissue function.