To quantitate calcium movements and membrane potential changes in stimulated neutrophils, we have measured net fluxes of Ca2+ and of the lipophilic cation tetraphenyl phosphonium by a very sensitive ion-selective electrode system. Activation of neutrophils by 3 X 10(-8) M phorbol 12-myristate, 13-acetate induces a release of approximately 20% of total cell calcium, with an initial lag period of less than 10 s. The Ca2+ outflux is markedly reduced in ATP-depleted cells and in the presence of a calmodulin inhibitor, thereby suggesting that it occurs by activation of the ATP-driven Ca2+ pump of the neutrophil plasmalemma. Activation of neutrophils also induces a transiently increased exchange of medium 45Ca with cell calcium, which is measurable a few seconds after cell exposure to the stimulant and peaks at approximately 40 s. Stimulation of neutrophils after attainment of steady-state accumulation of tetraphenyl phosphonium (resting potential of -67 mV) results in a marked depolarization, with a lag period of approximately 60 s. The rate and extent of depolarization are reduced by 40 and 65%, respectively, in a low Na+ medium but are not modified by an inhibitor of anion exchange across membranes. A high-K+ medium depolarizes neutrophils without either modifying their resting oxidative metabolism or impairing stimulability by the phorbol ester. Phorbol 12-myristate, which also exhibits no effect on the oxidative metabolism of neutrophils, does not induce Ca2+ extrusion and membrane potential changes. The causal relationship between Ca2+ mobilization, membrane potential changes and activation of neutrophil functions is discussed.