Endothelin (ET) receptors were identified in trout tissues that included the gill, heart, liver, kidney, and intestine. Since both the presence and the physiological function of endothelin-1 (ET-1) in trout gills have been determined, we examined the localization and characterization of ET receptors in the gills. Ligand specificity of the receptors, determined by using members of the endothelin family, revealed that endothelin receptors specific for ET-1 were predominant in the gills, but the binding sites of such receptors had low affinity for BQ-123, which is selective for mammalian endothelin A receptors (ET(A)). We propose that this subtype of receptor be called a fish-type ET(A) receptor (ET(AF)) to distinguish it from the mammalian ET(A) receptor, Affinity labeling of binding sites on gill membranes with I-125-labeled ET-1 and the cross-linking reagent 1,5-difluoro-2,4-dinitrobenzene revealed one major protein with an M(r) of 58,000, similar to that found in mammals, Autoradiographic studies demonstrated a high density of endothelin-binding sites in the lamellar sinusoid, which is involved in gas exchange in gills. No binding was detected in the chondrocytes or cartilaginous matrix. Our observations suggest that the endothelin and ET receptor system found in mammals might play a physiological role in the regulation of metabolic functions in fish.