Electrophysiological observations of neurons suggest that perikarya and dendrites differ in local intracellular Cl-concentration ([Cl-]i), that has not been demonstrated yet. Regional [Cl-]i in cultured hippocampal neurons was estimated using a Cl-sensitive fluorescent dye. Calibration showed that perikaryonic [Cl-]i was lower than dendritic [Cl-]i. Ethacrynic acid, an inhibitor of the outwardly directed Cl-pump, increased the perikaryonic but not dendritic [Cl-]i. A decrease in [Cl-]i induced by furosemide or bumetanide, inhibitors of Na+/K+/2Cl- cotransporters, was more prominent in dendrites than in perikaria. These findings suggest that uneven distribution of Cl- is generated by the region-specific localization of these transporters.