We have investigated the effect of external H+ concentration ( [H+](o)) on the human-ether-a-go-go-related gene (HERG) current (I-HERG), the molecular equivalent of the cardiac delayed rectifier potassium current (I-Kr), expressed in Xenopus oocytes, using the two-microelectrode voltage-clamp technique. When [H+](o) was increased, the amplitude of the I-HERG elicited by depolarization decreased, and the rate of current decay on repolarization was accelerated. The activation curve shifted to a more positive potential at lower external pH (pH(o)) values (the potential required for half-maximum activation, V-1/2, was: -41.8 mV, -38.0 mV, -33.7 mV, -26.7 mV in pH(o) 8.0, 7.0, 6.6, 6.2, respectively). The maximum conductance (g(max)) was also affected by [H+](o): a reduction of 7.9%, 14.6%, and 22.8% was effected by decreasing pH(o) from 8.0 to 7.0, 6.6, and 6.2, respectively. We then tested whether this pH effect was affected by the external Ca2+ concentration, which is also known to block HERG channels. When the extracellular Ca2+ concentration was increased from 0.5 mM to 5 mM, the shift in V-1/2 caused by increasing [H+](o) was attenuated, suggesting that these two ions compete for the same binding site. On the other hand, the decrease in g(max) caused by increasing [H+], was not significantly affected by changing external Ca2+ levels. The results indicate that HERG channels are inhibited by [H+](o) by two different mechanisms: voltage-dependent blockade (shift of V-1/2) and the decrease in g(max). With respect to the voltage-dependent blockade, the interaction between H+ and Ca2+ is competitive, whereas for the decreasing g(max) their interaction is non-competitive.