Loop diuretic-sensitive (Na+,K+,Cl-)-cotransport activity was found to be present in basolateral membrane vesicles of surface and crypt cells of rabbit distal colon epithelium. The presence of gradients of all three ions was essential for optimal transport activity. (Na+,K+) gradient-driven 36Cl fluxes were half-maximally inhibited by 0.14 microM bumetanide and 44 microM furosemide. While 86Rb uptake rates showed hyperbolic dependencies on Na+ and K+ concentrations with Hill coefficients of 0.8 and 0.9, respectively, uptakes were sigmoidally related to the Cl concentration, Hill coefficient 1.8, indicating a 1 Na+:1 K+:2 Cl stoichiometry of ion transport. The interaction of putative (Na+,K+,Cl-)-cotransport proteins with loop diuretics was studied from equilibrium-binding experiments using [3H]-bumetanide. The requirement for the simultaneous presence of Na+,K+, and Cl-, saturability, reversibility, and specificity for diuretics suggest specific binding to the (Na+,K+,Cl-)-cotransporter. [3H]-bumetanide recognizes a minimum of two classes of diuretic receptor sites, high-affinity (KD1 = 0.13 microM; Bmax1 = 6.4 pmol/mg of protein) and low-affinity (KD2 = 34 microM; Bmax2 = 153 pmol/mg of protein) sites. The specific binding to the high-affinity receptor was found to be linearly competitive with Cl- (Ki = 60 mM), whereas low-affinity sites seem to be unaffected by Cl-. We have shown that only high-affinity [3H]-bumetanide binding correlates with transport inhibition raising questions on the physiological significance of diuretic receptor site heterogeneity observed in rabbit distal colon epithelium.