We demonstrate binding of [125I][Nle13-po]motilin to homogenates of cat gastric and small intestinal, but not to colonic smooth muscle tissue. The density was (Bmax in fmol/mg protein): 0 (fundus); 12 +/- 2 (corpus); 22 +/- 3 (antrum); 55 +/- 12 (duodenum); 44 +/- 10 (jejunum); 17 +/- 1 (ileum); 0 (colon). A significant (p < 0.05) difference was found between the dissociation constant for motilin in the stomach (pKd = 8.84 +/- 0.06) and in the small intestine (pKd = 8.58 +/- 0.08). The motilides erythromycin-A (EM-A), EM-523, and EM-A N-oxide displaced labeled [Nle13-po]motilin bound to cat duodenal receptor with potencies (pKd) of 5.47 +/- 0.23, 7.60 +/- 0.24, and < 4.3, respectively. Studies with [Leu13-po]motilin fragments showed that the N-terminus of motilin interacts with the receptor. In the tissue bath, duodenal strips mounted in the longitudinal direction responded to motilin, EM-523, and EM-A (pEC50: 8.29 +/- 0.08; 7.12 +/- 0.12; 5.99 +/- 0.15). The compounds had a comparable intrinsic activity (83 +/- 3%; 80 +/- 5%; 82 +/- 5% of the response to ACh), which was unaffected by atropine, TTX, hexamethonium, and zacopride but reduced by verapamil and calcium-free medium. Cat stomach and small intestine possess smooth muscle motilin receptors, which have comparable properties as those found in man and in rabbit.