Human ether-à-go-go-related gene (HERG) encodes a K channel similar to the rapid delayed rectifier channel current (I_Kr) in cardiac myocytes. Modulation of I_Kr by extracellular acidosis under pathological conditions may impact on cardiac electrical activity. Therefore, we studied the effects of extracellular acidification on I_Kr function and the underlying mechanism, using HERG expressed in Xenopus oocytes as a model. Acidification [extracellular pH (pH_o) 8.5-6.5] accelerated HERG deactivation (at 80 mV, the time constant  of the major component of deactivation was 253 ± 17, 158 ± 10, and 65 ± 5 ms at pH_o 8.5, 7.5, and 6.5, respectively; n = 7-10 each), with no effects on other gating kinetics except a modest acceleration of recovery from inactivation (at 80 mV,  of recovery was 4.7 ± 0.3, 3.8 ± 0.3, and 1.3 ± 0.2 ms at pH_o 8.5, 7.5, and 6.5, respectively; n = 4-7 each). The following were ruled out as the underlying mechanisms: 1) voltage shift in channel activation, 2) pore blockade by protons, 3) protonation of histidines on the extracellular domain of HERG, 4) acceleration of recovery from C-type inactivation, and 5) interaction between an external H⁺ binding site and the cytoplasmic NH₂-terminal domain (a key determinant of HERG deactivation rate). Extracellular application of diethylpyrocarbonate caused an irreversible acceleration of HERG deactivation and prevented further acceleration by external acidification. Our data suggest that side chains accessible to the extracellular solution mediated the effects of elevating extracellular H⁺ concentration on channel deactivation.