Thiazides and related diuretics inhibit NaCl reabsorption in the distal tubule through an unknown mechanism. We report here that [3H]metolazone, a diuretic with a thiazide-like mechanism of action, labels a site in rat kidney membranes that has characteristics of the thiazide-sensitive ion transporter. [3H]Metolazone bound with high affinity (Kd = 4.27 nM) to a site with a density of 0.717 pmol/mg of protein in kidney membranes. The binding site was localized to the renal cortex, with little or no binding in other kidney regions and 11 other tissues. The affinities of thiazide-type diuretics for this binding site were significantly correlated with their clinical potency. Halide anions (Cl-, Br-, and I-) specifically inhibited high-affinity binding of [3H]metolazone to this site. [3H]Metolazone also bound with lower affinity (Kd = 289 nM) to sites present in kidney as well as in liver, testis, lung, brain, heart, and other tissues. Calcium antagonists and certain smooth muscle relaxants had Ki values of 0.6-10 microM for these low-affinity sites, which were not inhibited by most of the thiazide diuretics tested. Properties of the high-affinity [3H]metolazone binding site are consistent with its identity as the receptor for thiazide-type diuretics.