Motor effects of 13-norleucine motilin (13-Nle-M), a synthetic analog of motilin and biologically equivalent to the natural polypeptide, on the rabbit, guinea pig, rat, and human alimentary tract were investigated in vitro. Whereas guinea pig and rat preparations proved refractory to 13-Nle-M action, muscle strips of the stomach and upper small intestine from rabbits and man were highly sensitive to 13-Nle-M, contractile responses being elicited with concentrations of less than 2 times 10-minus 9 M. Although circular muscle from rabbit colon responded to 13-Nle-M, Taenia coli preparations were unaffected by the polypeptide; in man, the reverse was observed. Gallbladder, uterine, and vascular smooth muscle were unresponsive to 13-Nle-M. Pharmacological analysis revealed that the effects of 13-Nle-M on the gastrointestinal muscle are not mediated via nervous pathways: ganglion blockade by hexamethonium, blockade of axonal conduction by tetrodotoxin, or anticholinergic action by atropine failed to affect 13-Nle-M actions. It was therefore concluded that 13-Nle-M caused contractions by stimulating receptors on or in the muscle cell. By use of the antihistaminic pheniramine, histamine receptors could be differentiated from the site of 13-Nle-M action. As the contractile response to 13-Nle-M was abolished by the Ca++ antagonistic compound verapamil, a role for 13-Nle-M in the transport of Ca++ to the cytosol of intestinal smooth muscle might be considered.