Motor effects of graded concentrations of pure natural porcine motilin (13-Met-M) and synthetic motilin analogues - the methionine in position 13 substituted with either norleucine (13-Nle-M) or leucine (13-Leu-M) - on the rabbit, guinea-pig, rat, and human gastrointestinal smooth muscle were examined in vitro. Congruent species specificity of the motor activity of the motilins under study could be demonstrated in that muscle strips from rabbit and man were highly sensitive, whereas guinea-pig and rat preparations proved refractory to the polypeptides. In rabbit duodenal muscle and fundic muscle of the human stomach, graded concentrations of 13-Met-M, 13-Nle-M, and 13-Leu-M, respectively, produced graded increases in the contractile responses. The concentration-response curves were superimposable. Calculated maximal contractile responses (CMR's) and polypeptide doses for one-half maximal responses (D50 values) were not significantly different between the three motilins. Moreover, pharmacological analysis revealed that the motor effects of 13-Met-M, 13-Nle-M, and 13-Leu-M are uniformly not mediated via nervous pathways: neither blockage of axonal conduction by tetrodotoxin nor anticholinergic action by atropine exerted any detectable influence. Viewing the data presented, one may conclude that in man and rabbit, 1) natural porcine motilin and its synthetic position 13-substituted analogues, 13-Nle-M and 13-Leu-M, are of equal efficacy for gastroduodenal motor activity, and 2) position 13 of the amino acid sequence of motilin (22-residue chain) is not pertinent to the active site of the molecule.