The lung endothelium layer is exposed to continuous CO2 transit which exposes the endothelium to a substantial acid load that could be detrimental to cell function. The Na+/H+ exchanger and HCO3--dependent H+-transporting mechanisms regulate intracellular pH (pH(cyt)) in most cells. Cells that cope with high acid loads might require additional primary energy-dependent mechanisms. V-H+-ATPases localized at the plasma membranes (pmV-ATPases) have emerged as a novel pH regulatory system. We hypothesized that human lung microvascular endothelial (HLMVE) cells use pmV-ATPases, in addition to Na+/H+ exchanger and HCO3--based H+-transporting mechanisms, to maintain pH(cyt) homeostasis. Immunocytochemical studies revealed V-H+-ATPase at the plasma membrane, in addition to the predicted distribution in vacuolar compartments. Acid-loaded HLMVE cells exhibited proton fluxes in the absence of Na+ and HCO3- that were similar to those observed in the presence of either Na+, or Na+ and HCO3- The Na+- and HCO3--independent pH(cyt) recovery was inhibited by bafilomycin A(1), a V-H+-ATPase inhibitor. These studies show a Na+- and HCO3--independent pH(cyt) regulatory mechanism in HLMVE cells that is mediated by pmV-ATPases. (C) 2004 Elsevier Inc. All rights reserved.