Abstract  Voltage‐gated proton channels (H+ channels) are highly proton‐selective transmembrane pathways. Although the primary determinants for activation are the pH‐ and voltage‐gradients across the membrane, the current amplitudes fluctuate often when these gradients are constant. The aim of this study was to investigate the role of the intracellular pH (pHi) in regulating the availability of H+ channels in osteoclasts and microglia. In whole‐cell clamp recordings, the pHi was elevated after exposure to NH4Cl and returned to the control level after washout. However, the H+ channel conductance did not recover fully when the exposure was prolonged (>5 min). Similar results were observed in osteoclasts and microglia, but not in COS7 cells expressing a murine H+ channel gene (mVSOP). As other electrophysiological properties, like the gating kinetics and voltage‐dependence for activation, were unchanged, the decreases in the H+ channel conductance were likely due to the decreases in H+ channels available at the plasma membrane. The decreases in the H+ channel conductances were accompanied by reductions in the cell capacitance. Exposure to NH4Cl increased the uptake of the endocytosis marker, FM1–43, substantiating the idea that pHi increases facilitated endocytosis. In osteoclasts, whose plasma membrane expresses V‐ATPases and H+ channels, pHi increases by these H+‐transferring molecules in part facilitated endocytosis. The endocytosis and decreases in the H+ channel conductance were reduced by dynasore, a dynamin blocker. These results suggest that pHi increases in osteoclasts and microglia decrease the numbers of H+ channels available at the plasma membrane through facilitation of dynamin‐dependent endocytosis. This article is protected by copyright. All rights reserved