To combat bacterial infection, phagocytes generate superoxide (O2-) and other microbicidal oxygen radicals. NADPH oxidase, the enzyme responsible for O2- synthesis, is deficient in chronic granulomatous disease (CGD) patients. Although O2- generation is accompanied by a large burst of metabolic acid production, intracellular pH (pHi) remains near neutrality due to the concomitant stimulation of H+ extrusion. Three major pathways contribute to pHi regulation in activated phagocytes: Na+/H+ exchange, vacuolar-type H+ pumps, and a H+ conductance. The present study analyzed the relationship between activation of the NADPH oxidase and stimulation of the H+ extrusion mechanisms in human blood neutrophils. Phorbol ester-induced activation of Na+/H+ exchange and H+ pumping occurred normally in cells from CGD patients. Unlike normal individuals, however, CGD patients were unable to activate the H+ conductive pathway. Thus, activation of the H+ conductance appears to be contingent on the assembly of a functional NADPH oxidase. These findings imply a dual role of the NADPH oxidase in O2- synthesis and in the regulation of pHi. The oxidase (or some components thereof) may itself undertake H+ translocation or, alternatively, may signal the activation of a separate H+ conducting entity.