The rate of metabolic acid generation by neutrophils increases greatly when they are activated. Intracellular acidification is prevented in part by Na+/H+ exchange, but a sizable component of H+ extrusion persists in the nominal absence of Na+ and HCO3-. In this report we determined the contribution to H+ extrusion of a putative H+ conductive pathway and its mode of activation. In unstimulated cells, H+ conductance was found to be low and unaffected by depolarization. An experimental system was designed to minimize the metabolic acid generation and membrane potential changes associated with neutrophil activation. By using this system, beta-phorbol esters were shown to increase the H+ (equivalent) permeability of the plasma membrane. The direction of the phorbol ester-induced fluxes was dictated by the electrochemical H+ gradient. Moreover, the parallel migration of a counterion through a rheogenic pathway was necessary for the displacement of measurable amounts of H+ equivalents across the membrane. These findings suggest that the H+ flux is conductive. The effect of beta-phorbol esters was mimicked by diacylglycerol and mezerein and was blocked by staurosporine, whereas alpha-phorbol esters were ineffective. Together, these findings indicate that stimulation of protein kinase C induces the activation of an H+ conductance in the plasma membrane of human neutrophils. Preliminary evidence for activation of a separate, bafilomycin A1-sensitive H+ extrusion mechanism, likely a vacuolar type H(+)-ATPase, is also presented.