The effects of several sulfamoyl benzoic acid derivatives on Na−K−Cl cotransport were investigated in winter flounder intestine. The relative efficacy (IC50values) and order of potency of these derivatives were benzmetanide, 5×10−8m>bumetanide 3×10−7m>piretanide 3×10−6m>furosemide 7×10−6m>amino piretanide 1×10−53-amino-4-penoxy-5-sulfamoyl benzoic acid. Binding of [3H] bumetanide was studied in microsomal membranes from winter flounder intestine and compared to that in bovine kidney outer medulla. Binding was also studied in brush-border membranes from winter flounder intestine. The estimated values forKdand number of binding sites (n) were: bovine kidney,Kd=1.6×10−7,n=10.5 pmol/mg protein; winter flounder intestine,Kd1.2×10−7,n=7.3 pmol/mg protein, and brush-border membranes from winter flounder,Kd=5.3×10−7,n=20.4 pmol/mg protein. The estimatedKdfor bumetamide binding to winter flounder brush-border membranes derived from association and dissociation kinetics was 6.8×10−7m. The similarity in magnitudes of IC50andKdfor bumetanide suggests that the brush-border cotransporter is ordinarily rate-limiting for transmural salt absorption and that bumetanide specifically binds to the cotransporter. Measurement of bumetanide binding at various concentrations of Na, K and Cl showed that optimal binding required all three ions to be present at about 5mmconcentrations. Higher Na and K concentrations did not diminish binding but higher Cl concentrations (up to 100mmCl) inhibited bumetanide binding by as much as 50%. Still higher Cl concentrations (500 and 900mm) did not further inhibit bumetanide binding. Scatchard analysis of bumetanide binding at 5 and 100mmCl concentrations showed that bothKdandnwere lower at the higher Cl concentration (5mmCl:Kd=5.29×10−7m,n=20.4 pmol/mg protein; 100mmCl:Kd=2.3×10−7m,n=8.8 pmol/mg protein). These data suggest two possibilities: that bumetanide and Cl binding are not mutually exclusive (in contrast to pure competitive inhibition) and that they each bind to separate sites or that two distinct bumetanide binding sites exist, only one of which exhibits Cl inhibition of binding. This inhibition would then be consistent wi