Aqueous humor secretion is in part linked to transport by nonpigmented ciliary epithelium (NPE) cells. During this process, the cells must maintain stable cytoplasmic pH (pH_i). Because a recent report suggests that NPE cells have a plasma membrane-localized vacuolar H⁺-ATPase, the present study was conducted to examine whether vacuolar H⁺-ATPase contributes to pH_i regulation in a rabbit NPE cell line. Western blot confirmed vacuolar H⁺-ATPase expression as judged by H⁺-ATPase 31-kDa immunoreactive polypeptide in both cultured NPE and native ciliary epithelium. pH_i was measured using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Exposing cultured NPE to K⁺-rich solution caused a pH_i increase we interpret as depolarization-induced alkalinization. Alkalinization was also caused by ouabain or BaCl₂. Bafilomycin A₁ (0.1 µM; an inhibitor of vacuolar H⁺-ATPase) inhibited the pH_i increase caused by high K⁺. The pH_i increase was also inhibited by angiotensin II and the metabolic uncoupler carbonyl cyanide m-chlorophenylhydazone but not by ZnCl₂, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), omeprazole, low-Cl medium, -free medium, or Na⁺-free medium. Bafilomycin A₁ slowed the pH_i increase after an NH₄Cl (10 mM) prepulse. However, no detectable pH_i change was observed in cells exposed to bafilomycin A₁ under control conditions. These studies suggest that vacuolar H⁺-ATPase is activated by cytoplasmic acidification and by reduction of the proton electrochemical gradient across the plasma membrane. We speculate that the mechanism might contribute to maintenance of acid-base balance in NPE.