Over 100 motilin fragments and analogues, including monosubstituted C-terminal-deleted analogues, conformationally restricted analogues, analogues with peptide bond isoteres (CH2-NH), and N-terminal fragments adjunct to an amphiphilic helix, were synthesized by solid-phase methodology, purified by reverse-phase HPLC, and assayed in vitro in a muscle strip bioassay (rabbit duodenum) and in a radioligand binding assay on crude membrane extracts from smooth muscle (rabbit antrum). The data suggest the existence of three distinct regions involved in the interaction of motilin with its receptor: the N-terminal region (amino acids 1-7) constitutes the minimal basic unit of binding and activity; the transition region (amino acidsa 8-9) links the N-terminal and C-terminal regions; and the C-terminal region (amino acids 10-22) forms an alpha-helix that stabilizes the interaction of the N-terminal residues at the active site.