The ionic basis of phorbol 12-myristate 13-acetate (PMA)-stimulated membrane depolarization in the human neutrophil has not previously been established. Alterations in cation permeability are probably not directly responsible for the depolarization response, since the rate or Rb+ influx or efflux is unchanged upon PMA stimulation, and although Na+ fluxes are increased, depolarization is not changed by either the addition of ouabain or reduction of extracellular Na+ from 140 to 0 meq. Furthermore, the enhanced Na+ influx in stimulated cells is blocked by amiloride at 10(-3) M, but not by 10(-5) M, suggesting Na+ influx proceeds through the electroneutral Na(+)-H+ antiporter and is therefore not responsible for depolarization. Upon stimulation, Cl- content of PMA-stimulated neutrophils decreases without change in Na+ or K+ content, as determined by electron probe analysis. In addition, acute reduction in extracellular Cl- enhances the rate and extent of depolarization induced by PMA. This change in intracellular Cl- and effect of reduction in extracellular Cl- concentration on depolarization can best be accounted for by an enhanced efflux via an electrogenic mechanism. Thus enhanced conductive Cl- efflux can account for the observed depolarization. That Ca2+ regulates depolarization is evidenced by the dependence of depolarization on external Ca2+ (Cao2+). Depolarization is absent in Ca2(+)-depleted cells [internal Ca2+ (Cai2+) less than 15 nM] and is restored with titration of extracellular Ca2+, exhibiting a 50% effective dose (ED50) of 100 mM. Thus PMA-initiated depolarization is regulated by Ca2+, either from intra- or extracellular sources, but the Ca2(+)-dependent activity responsible for control of Cl- efflux is as yet uncharacterized.