HERG (Human ether-à-go-go-related gene) potassium channels are crucial for cardiac action potential repolarization. HERG channels are also found in neuronal and tumor cells. The effect of pHo on HERG is of clinical significance because of changes in pH during myocardial ischemia, inflammation, and respiratory alkalosis. We present evidence for the presence of multiple proton binding sites in HERG. Extracellular protons bind rapidly and reversibly to affect both activation and deactivation. However, these effects occur in two distinct pHo ranges. The deactivation rate has a pKa of 6.76 +/- 0.02 compared to pKa of 5.50 +/- 0.02 for changes in current suppression, which suggests the presence of at least two proton binding sites on HERG with functionally distinct properties.