Cl(-)-stimulated and ethacrynic acid-sensitive ATPase (Cl(-)-ATPase) of plasma membrane origin in the rat brain is a candidate for an active outwardly directed Cl- translocating system. Biochemistry of Cl(-)-ATPase and ATP-dependent Cl- transport (Km values for ATP and Cl-, nucleotide specificity, pH dependency, and sensitivity to ethacrynic acid) suggested that Cl(-)-ATPase is an ATP-driven Cl- pump. Activity of the reconstituted Cl(-)-ATPase/pump increased in the presence of phosphatidylinositol-4-monophosphate, and this pump activity further increased at an inside-positive membrane potential or in the presence of a protonophore, suggesting that the Cl(-)-ATPase/pump is an electrogenic Cl- transporter, probably regulated by phosphoinositide turnover in vivo. In cultured hippocampal pyramidal cell-like neurons from embryonic rat brain, ethacrynic acid and ATP-consuming treatment increased, but furosemide, an inhibitor of Na+/K+/Cl- cotransporter, decreased, [Cl-]i when monitored using Cl(-)-sensitive fluorescent probes. The stationary levels of [Cl-]i were lower and the effects of ethacrynic acid were more prominent in perikarya than in dendrites, while the effects of furosemide were more obvious in dendrites than in perikarya. The lower perikaryonic [Cl-]i and the marked effects of ethacrynic acid were observed in the later stage rather than in the early stage of culture. Thus, region-specific localization and developmental changes in the activities of Cl- transporters probably result in uneven and age-dependent distribution of Cl- in the neurons.