A plasma membrane proton-translocating adenosinetriphosphatase (ATPase) has been identified in rat alveolar pneumocytes in primary culture using the pH-sensitive fluorescent probe 2',7'-biscarboxyethyl-5,6-carboxyfluorescein. Intracellular pH (pHi) was acutely lowered by NH3 prepulse in HCO3(-)-free medium buffered with 6 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, and its recovery was measured thereafter under control conditions, in the presence of amiloride to inhibit Na(+)-H+ antiport, and in the presence of N-ethylmaleimide (NEM), a plasma membrane H(+)-ATPase inhibitor. Initial rate of pHi recovery was reduced by 67% in the presence of amiloride, 52% in the presence of NEM, and 96% in the presence of both. Recovery was decreased but not abolished in Na(+)-free buffer, was essentially abolished when NEM was present in the absence of Na+, and was also abolished by addition of the metabolic inhibitor KCN in glucose- and Na(+)-free medium. These data suggest that alveolar epithelial cells possess a plasma membrane H(+)-ATPase. In Na(+)-containing buffer at pH 7.4, steady-state pHi was 7.50. This value was unaffected by amiloride but decreased to 7.01 in the presence of NEM, suggesting active H(+)-ATPase and inactive Na(+)-H+ antiport at steady-state pHi. We conclude that this plasma membrane proton-translocating ATPase in alveolar pneumocytes may be an important mechanism contributing to regulation of steady-state pHi, recovery from acute intracellular acidification, and modulation of extracellular alveolar fluid pH.