T he relationship of intracellular pH (pHi) to superoxide radical (O2-) generation was investigated in chemotactic factor-stimulated human neutrophils. Exposure of cells to 100 nM N-formylmethionyl-leucyl-phenylalanine (FMLP) caused activation of Na/H exchange which, in 140 mM Na medium (pH0 7.40), led to a rise in pHi from 7.22 to 7.80. This pHi change was sensitive to amiloride (apparent Ki 78 microM), an inhibitor of Na/H countertransport. The time course of the alkalinization was similar to that of FMLP-stimulated O2- production, which was complete by 5 min. In the presence of 1 mM amiloride, which nearly blocked the pHi transient elicited by FMLP, or in the absence of external Na, where intracellular acidification was observed in FMLP-stimulated cells, O2- release was still roughly 25-45% of normal. Thus, an alkalinization cannot be an obligatory requirement for O2- generation. By independently varying either pH0, pHi, or the internal or external concentrations of Na, both the direction and magnitude of the FMLP-induced pHi transients could be altered. In each instance, the amount of O2- release correlated directly with pHi and was enhanced by intracellular alkalinization. In the absence of FMLP, a rise in pHi to 7.7-7.8 by exposure of cells to 30 mM NH4Cl, 10 microM monensin (a Na/H exchanging ionophore), or after a prepulse with 18% CO2 did not result in O2- generation. Thus, these results imply that an alkalinization per se is not a sufficient trigger. Neutrophils exposed to 4 nM FMLP exhibited a threefold slower rate of alkalinization (reaching pHi approximately 7.80 by 20-30 min) as compared to that obtained with 100 nM FMLP and did not release significant amounts of O2- under normal incubation conditions. However, these cells could be induced to generate O2- when the degree of alkalinization was enhanced by internal Na depletion or by pretreatment with 18% CO2. Together, these results indicate a modulating effect of pHi on O2- production and suggest that other functional responses of neutrophils may be regulated by their pHi.