Abstract: Objective To investigate the effects of motilin agonists on intracellular Ca2+ mobilization in primary cultured rat myenteric neurons. Methods Motilin-induced and erythromycin-induced intracellular Ca2+ signaling was studied in primary cultures of rat myenteric neurons using the radiometric Ca2+ indicator Furo3/AM with a laser confocal microscope. Results In Hank’s solution, 10-8, 10-7, and 10-6 mol/L motilin could elevate intracellular Ca2+ concentration ([Ca2+]i) to the peak levels of 10.6±2.1, 15.9±1.2, and 30.6±3.7 respectively with their relative percentage change in fluorescent intensity of (40.1±6.3)%, (63.0±11.2)%, and (100.8±18.4)% respectively, indicating the dose-dependent effect of motilin on [Ca2+]i. In Hank’s solution, 10 μg/ml erythromycin could induce the elevation of [Ca2+]i to the average peak of 23.2±5.6 with the relative percentage change in fluorescent intensity of (82.8±13.0)%. When pretreated with the antibody against motilin receptor in Hank’s solution, the effect of 10 μg/ml erythromycin was almost inhibited completely. Conclusion Motilin can increase [Ca2+]i, and erythromycin also has this effect by binding to motilin receptor.