An Na(+)- and HCO3(-)-independent mechanism of cytoplasmic pH (pHi) recovery was previously demonstrated in acid-loaded macrophages (Swallow, C. J., Grinstein, S., and Rotstein, O. D. (1988) J. Biol. Chem. 263, 19558-19563). Acid extrusion was found to be ATP-dependent and sensitive to N-ethylmaleimide and N,N'-dicyclohexylcarbodiimide, suggesting involvement of an H(+)-pumping ATPase. In this report, the properties and mode of activation of this putative pump were studied in detail. In acid-loaded cells, pHi recovery, measured using a fluorescent probe, was found to be insensitive to azide or oligomycin, which are inhibitors of F0F1 (mitochondrial) H(+)-ATPases, and to vanadate, an inhibitor of E1E2-type ATPases. Instead, the recovery was sensitive to the vacuolar type H(+)-ATPase inhibitors 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, p-chloromercuribenzenesulfonic acid, and bafilomycin A1. Using the fluorescent probes bisoxonol and 3,3'-dipropylthiodicarbocyanide iodide to measure the membrane potential of intact cells, acid loading of macrophages was shown to result in an N,N'-dicyclohexylcarbodiimide-sensitive hyperpolarization of approximately 15 mV. This hyperpolarization was not inhibited by charybdotoxin, suggesting that it was not due to efflux of K+ through Ca2(+)-activated K+ channels, but may instead be due to electrogenic pumping of protons across the plasma membrane. This was consistent with the partial dependence of the Na(+)- and HCO3(-)-independent pHi recovery on the presence of intracellular Cl-. As in vacuolar membranes, Cl- appears to act as a counterion to H+, preserving electroneutrality and thus facilitating pHi recovery. In acid-loaded urinary epithelial cells, activation of H+ pumping occurs by exocytic insertion of intracellular (vacuolar) H(+)-ATPases into the plasma membrane. In this system, exocytosis is triggered by an associated increase in the cytoplasmic free Ca2+ concentration and is microtubule-dependent. We determined whether an analogous process exists in macrophages. Acid loading of macrophages induced an approximately 120 nM increase in cytoplasmic free Ca2+ concentration due to mobilization of Ca2+ from an intracellular source. However, preventing this increase by preloading macrophages with bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid did not inhibit the Na+ and HCO3(-)-independent pHi recovery, neither was the recovery inhibited by microtubular disruption using 0.1 mM colchicine. Furthermore, cytoplasmic acid loading did not cause a detectable release of secretory granular, endosomal, or lysosomal contents, suggesting that activation of H+ pumping at the cell surface is not mediated by exocytic fusion of these compartments with the plasma membrane. Taken together, these data suggest that H(+)-ATPases are constitutively present in the macrophage plasma membrane.(ABSTRACT TRUNCATED AT 400 WORDS)