Alkalinization of the cytoplasm in human fibroblasts activates Cl- efflux. The pathway of Cl- efflux is inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) or by removal of Cl- from the extracellular medium, but it is insensitive to the Cl(-)-cation cotransport inhibitor bumetanide. These data indicate that the alkali-stimulated Cl- pathway is a Cl(-)-anion exchanger. One function of this pathway appears to involve intracellular pH homeostasis. Thus, after alkalinization of the cytoplasm, recovery of the pH to basal levels is impaired by DIDS or by removal of HCO3- from the extracellular medium, but it is unaffected by the Na(+)-H+ exchange inhibitor amiloride. Reports from a number of laboratories, including our own, have described a biphasic efflux of Cl- in which kinetics suggested the presence of two subcellular Cl- compartments. The data presented in this study provide a mechanism to explain this kinetic behavior. The biphasic Cl- efflux kinetics are the result of a transient alkalinization of the cytoplasm associated with a change in PCO2 as cells are transferred from the incubator to the bench top for the efflux assay.