The use of the distally-acting diuretic, chlorothiazide, has been reported to have important effects on renal magnesium handling. The cellular mechanisms of chlorothiazide action on Mg2+ uptake was investigated in immortalized mouse distal convoluted tubule (MDCT) cells. Intracellular free Mg2+ concentration was determined by microfluorescence. Mg2+ transport was measured as a function of change in intracellular Mg2+ concentration with time following placement of Mg2+-depleted cells into a buffer containing 1.5 mM magnesium. The uptake rate of Mg2+ into Mg2+-depleted cells was 179 +/- 28 nM/second. Mg2+ uptake was dependent on the membrane voltage as membrane hyperpolarization enhanced uptake whereas depolarization diminished transport. Chlorothiazide increased Mg2+ uptake by 58%, from 179 +/- 28 to 283 +/- 23 nM/second. The ability of chlorothiazide to stimulate Mg2+ uptake in MDCT cells was concentration-dependent and related to the diuretic-induced hyperpolarization of the plasma membrane. These studies support the notion that acute chlorothiazide administration enhances renal magnesium conservation through its effects on Mg2+ transport within the distal convoluted tubule. Since chronic chlorothiazide administration may result in hypokalemia as well as hypomagnesemia, Mg2+ uptake was determined in potassium-depleted MDCT cells. Mg2+ uptake was diminished, 80 +/- 24 nM/second, in potassium depleted cells. Hyperpolarization of the plasma membrane with the cell permanent anion, SCN-, corrected Mg2+ uptake in potassium depleted cells suggesting that the basis for diminished uptake may, in part, be due to depolarization of the membrane voltage. In summary, acute chlorothiazide stimulates Mg2+ transport in MDCT cells. We postulate that chronic chlorothiazide use may lead to hypokalemia that in turn diminishes Mg2+ transport in the distal tubule resulting in urinary magnesium-wasting.