Natural porcine motilin (13-methionine-motilin) and its synthetic position 13-substituted analogues, 13-norleucine-motilin and 13-leucine-motilin, are of equal efficacy for gastrointestinal motor activity. Pharmacological in vitro analysis reveals that motilin effects on the gastrointestinal muscle are not mediated via nervous pathways but are brought about by direct action of the polypeptide on the muscle cell. As the contractile response to motilin can be abolished by the Ca++ antagonistic compound verapamil, a role for motilin in the transport of Ca++ to the cytosol of intestinal smooth muscle might be considered. Ca++ fluxes seem to be linked to intracellular cyclic guanosine-3':5'-monophosphate and the antagonistic cyclic adenosine-3':5'-monophosphate. By contrast, in motilin-stimulated gastric pepsin secretion, there is no evidence for a mediating role of cyclic nucleotides. It is more likely that the pepsigogic effect is brought about by augmented gastric mucosal blood flow.