Motilin receptors in rabbit antral and duodenal smooth muscle tissue were characterized by direct binding technique using 125I-labeled porcine motilin as a tracer ligand. Binding at 30 degrees C was maximal at 90 min, was saturable and partially reversible. Displacement studies with natural porcine motilin, synthetic leucine-motilin or norleucine-motilin indicated a dissociation constant (Kd) of 1.1 +/- 0.3 nM and a maximal binding capacity (Bmax) of 42 +/- 10 fmol/mg protein. Binding was unaffected by glucagon, pancreatic polypeptide and somatostatin, but substance P interfered via an unknown mechanism. By density gradient centrifugation motilin receptors were shown to be present in plasma membranes. Binding could only be demonstrated in preparations from antrum and upper duodenum. These observations provide evidence for a localized target region for motilin in the gastrointestinal tract, and for a direct interaction of motilin with gastrointestinal smooth muscle tissue.