The present study was undertaken to explore motilin's transduction pathway in the rabbit, Guanine nucleotides inhibited I-125-motilin binding in rabbit antral tissue and increased the dissociation of motilin from its receptor. Motilin, the motilin agonist erythromycin A enol ether (EM-201) and carbachol (taken as control) increased the production of inositol phosphates in rabbit duodenal smooth muscle strips labeled with myo-[2-H-3]inositol. The effect of carbachol was blocked by atropine, Dose-response curves revealed that 50% of this effect was obtained with 3.9 nM motilin, 170 nM EM-201, 0.54 mu M carbachol. Chromatographic separation of the inositol phosphate metabolites showed significant increases in the levels of [H-3]inositol bisphosphate and of [H-3]inositol trisphosphate. The three substances were without effect upon the metabolism of cAMP, nor did they modulate the rise in cAMP induced by GTP. We propose that motilin's transduction pathway uses a G protein that causes an increase in inositol trisphosphate which is rapidly metabolized, and which may release calcium from intracellular stores.