Melatonin, a close derivative of serotonin, is involved in physiological regulation of circadian rhythms. In the gastrointestinal (GI) system, melatonin exhibits endocrine, paracrine and autocrine actions and is implicated in the regulation of GI motility. Generally, melatonin actions oppose the stimulatory actions of serotonin on motility. However, it is not known whether melatonin can also act directly on GI smooth muscle cells.AIMTo determine the expression of melatonin receptors in smooth muscle and identify their signaling pathways.METHODSMuscle cells were isolated from rabbit stomach by enzymatic digestion. Expression of melatonin receptors, MT1 and MT2, was determined by RT‐PCR and Western blot. G protein activity was measured by melatonin‐induced increase in Gα binding to [35S]GTPγS. PLC‐β activity was measured by ion‐exchange chromatography. Cytosolic Ca2+ was measured in fura‐2 loaded cells and muscle contraction by scanning micrometry.RESULTSMT1 was detected by RT‐PCR and western blot. Melatonin activated Gαq, but not Gαs, Gαi1, Gαi2, or Gαi3. Consistent with activation of Gαq, melatonin stimulated PI hydrolysis, increased cytosolic Ca2+, and elicited muscle contraction.CONCLUSION. Gastric smooth muscle cells express receptors for melatonin (MT1) coupled to Gq. The receptors mediate stimulation of PLC‐β activity and increase in cytosolic Ca2+, and elicit muscle contraction.