Thiazide diuretics inhibit Na+ and stimulate Ca2+ absorption in renal distal convoluted tubules. Experiments were performed on immortalized mouse distal convoluted tubule (MDCT) cells to determine the mechanism underlying the dissociation of sodium from calcium transport and the stimulation of calcium absorption induced by thiazide diuretics. Control rates of 22Na+ uptake averaged 272 +/- 35 nmol min-1 mg protein-1 and were inhibited 40% by chlorothiazide (CTZ, 10(-4) M). Control rates of 36Cl- uptake averaged 340 +/- 50 nmol min-1 mg protein-1 and were inhibited 50% by CTZ. CTZ stimulated 45Ca2+ uptake by 45% from resting levels of 2.86 +/- 0.26 nmol min-1 mg protein-1. Bumetanide (10(-4) M) had no effect on 22Na+, 36Cl-, or 45Ca2+ uptake. Control levels of intracellular calcium activity ([Ca2+]i) averaged 91 +/- 12 nM. CTZ elicited concentration-dependent increases of [Ca2+]i to a maximum of 654 +/- 31 nM at 10(-4) M. CTZ reduced intracellular chloride activity ([Cl-]i), as determined with the chloride-sensitive fluorescent dye 6-methoxy-N-(3-sulfopropyl)quinolinium. The chloride channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 10(-5) M) abolished the effect of CTZ on [Cl-]i. NPPB also blocked CTZ-induced increases of 45Ca2+. Resting membrane voltage, measured in cells loaded with the potential-sensitive dye 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)], averaged -72 +/- 2 mV. CTZ hyperpolarized cells in a concentration-dependent and reversible manner. At 10(-4) M, CTZ hyperpolarized MDCT cells by 20.4 +/- 7.2 mV. Reduction of extracellular Cl- or addition of NPPB abolished CTZ-induced hyperpolarization. Direct membrane hyperpolarization increased 45Ca2+ uptake whereas depolarization inhibited 45Ca2+ uptake. CTZ-stimulated 45Ca2+ uptake was inhibited by the Ca2+ channel blocker nifedipine (10(-5) M). We conclude that thiazide diuretics block cellular chloride entry mediated by apical membrane NaCl cotransport. Intracellular chloride, which under control conditions is above its equilibrium value, exits the cell through NPPB-sensitive chloride channels. This decrease of intracellular chloride hyperpolarizes MDCT cells and stimulates Ca2+ entry by apical membrane, dihydropyridine-sensitive Ca2+ channels.