The effects of several sulfamoyl benzoic acid derivatives on Na-K-Cl cotransport were investigated in winter flounder intestine. The relative efficacy (IC50 values) and order of potency of these derivatives were benzmetanide, 5 X 10(-8) M greater than bumetanide 3 X 10(-7) M greater than piretanide 3 X 10(-6) M greater than furosemide 7 X 10(-6) M greater than amino piretanide 1 X 10(-5) 3-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 for Kd and number of binding sites (n) were: bovine kidney, Kd = 1.6 X 10(-7), n = 10.5 pmol/mg protein; winter flounder intestine, Kd 1.2 X 10(-7), n = 7.3 pmol/mg protein, and brush-border membranes from winter flounder, Kd = 5.3 X 10(-7), n = 20.4 pmol/mg protein. The estimated Kd for bumetanide binding to winter flounder brush-border membranes derived from association and dissociation kinetics was 6.8 X 10(-7) M. The similarity in magnitudes of IC50 and Kd for 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 5 mM concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)