Erythromycin A (EMA) is a potent stimulator of gastrointestinal motor activity. In vitro studies suggest that it mimics motilin, a peptide that stimulates motor activity in human and in rabbit via smooth muscle receptors. We have compared the in vitro contractile effect of EMA and two derivatives, 8,9-anhydro-EMA-6,9-hemiketal (EM201) and EMA N-oxide, on rabbit duodenal smooth-muscle strips with their ability to displace iodinated motilin bound to crude smooth-muscle membrane fractions. The concentrations required to induce 50% of the maximum contractile response to a supramaximal dose of acetylcholine were 5.0 x 10(-8), 2.0 x 10(-6), and 1.0 x 10(-4) M for, respectively, EM201, EMA, and EMA N-oxide. The concentrations required to displace 50% of the labeled motilin were, in the same order, 1.0 x 10(-8), 1.3 x 10(-7), and 4.0 x 10(-6) M. Both parameters were well correlated. The dose-response curve of the EMA was parallel to that of motilin and the effects of motilin and EMA were additive. Contractions induced by EMA were insensitive to pretreatment with tetrodotoxin or atropine. EMA had no effect on muscle strips of rat or dog duodenum but did induce contractions in human strips. EMA was totally ineffective on ileal preparations, which are also unresponsive to motilin and in which motilin binding is absent. EMA has therefore the same regional and species specificity as motilin. We conclude that EMA is a motilin receptor agonist.