Pharmacological studies indicate that in man and in rabbit, but not in dog, motilin has a direct influence upon gastrointestinal smooth muscle. In accordance with this hypothesis we have presented direct biochemical evidence for the presence of motilin receptors on rabbit smooth muscle tissue. We have now extended our studies to human and canine tissue. Tissue homogenates were studied in binding experiments with iodinated porcine [Leu13]motilin and iodinated canine motilin. It was ascertained that the iodination procedure had little effect on the biological activity of the porcine analogue. In the human antrum specific binding of the iodinated porcine analogue was only found in the smooth muscle layer. It was absent in mucosal or serosal preparations. At 30 degrees C and pH 8.0, binding was maximal after 60 min of incubation, and was reversed by the addition of unlabeled porcine motilin. Binding was enhanced in the presence of calcium and magnesium ions. At a concentration of 10 mM MgCl2, binding was 220% of the binding observed in its absence. Displacement studies with synthetic porcine [Leu13]motilin or synthetic natural porcine motilin indicated a dissociation constant (Kd) of 3.6 +/- 1.6 nM and a maximal binding capacity (Bmax) of 77 +/- 9 fmol per mg protein. Canine motilin displaced iodinated porcine motilin with an apparent Kd of 2.2 +/- 0.9 nM. Compared to antral binding, receptor density decreased aborally and orally, and was absent in jejunum and ileum. In dog specific binding could not be demonstrated in antral and duodenal tissue, neither with labeled porcine nor with labeled canine motilin. However, labeled canine motilin was equipotent to labeled porcine motilin in binding studies with human tissue: the dissociation constant was 0.9 +/- 0.6 nM. The present studies therefore demonstrate the existence of a specific motilin receptor in the antroduodenal region of the human gut. Apparently, such receptors are not present in the canine gut. Our data support the hypothesis that in the human gastrointestinal tract, the gastroduodenal area is motilin's target region.