Two distinct mechanisms have been shown to mediate cytoplasmic pH (pHi) recovery in acid-loaded peritoneal macrophages (M phi s): Na+/H+ exchange and H+ extrusion by vacuolar-type (V-type) H+ ATPases. The present studies examined the relative roles of these two systems in maintaining pHi and cell function. Measurements of M phi pHi and superoxide (O2-) production in response to stimulation with 12-O-tetradecanoyl phorbol 13-acetate (TPA) were made at physiological or acidic extracellular pH (pHo) levels. The V-type H+ ATPase inhibitor, bafilomycin A1, and the potent Na+/H+ exchange inhibitor, N-ethyl-N-propylamino amiloride (EPA), were used to examine the contributions of these ion transporters to pHi regulation and cell function. At pHo 7.35, the complementary activities of the Na+/H+ antiport and the V-type H+ ATPase mediate pHi homeostasis. At pHo 6.7, maintenance of pHi depends primarily on H+ ATPase activity: bafilomycin A1 reduced pHi from 6.8 +/- 0.02 in control cells to 6.59 +/- 0.01 (P < 0.01) while EPA was without effect. The functional importance of V-type H+ ATPase-activity in preserving pHi homeostasis at acidic extracellular pH levels was reflected by the impairment of O2- production at pHo 6.70 when H+ ATPase activity was inhibited: bafilomycin A1 reduced O2- production from 13.9 +/- 1.0 to 9.3 +/- 0.6 nmoles/10(6) cells/40 min, in control and bafilomycin A1-treated cells, respectively (P < 0.05), while EPA had no effect. In subsequent studies, pHi was independently manipulated using the ionophore nigericin. Lowering pHi from 6.80 to 6.60 reduced O2- production from 15.3 +/- 1.8 to 9.8 +/- 1.6 nmoles/10(6) cells/40 min (P < 0.05), indicating that the cytoplasmic acidification resulting from inhibition of H+ ATPases at low pHo could account for the associated impairment of O2- production. In a more profoundly acidic environment (pHo 6.35), H+ ATPases remained active in regulating pHi, but could not preserve a sufficiently physiological pHi to support respiratory burst activity. V-type H+ ATPases constitute the dominant mechanism by which the pHi of peritoneal M phi s is maintained in an acidic extracellular environment.