Numerous previous studies have demonstrated that ghrelin promotes gastric motility when administered peripherally. This effect appears to be regulatory but not directly stimulatory, and therefore may involve a number of complex mechanisms. In the periphery, ghrelin may affect gastric motility through intercellular networks among interstitial cells of Cajal, myenteric nerve cells and smooth muscle cells. The aim of the present study was to investigate the effects and possible mechanisms underlying this hypothesis. The effects of ghrelin on the contraction force of gastric antrum smooth muscle strips of rats were studied in the presence or absence of carbachol (CCh), [D-Lys3]-GHRP-6, atropine, tetrodotoxin (TTX) and nimodipine in vitro. The expression of ghrelin receptors (GHS-Rs) on different cell types in gastric muscle layers was observed by means of immunofluorescence. Ghrelin enhanced smooth muscle strip contraction induced by CCh, but when CCh was absent; this effect was eliminated. Atropine and nimodipine eradicated the muscle strip contraction enhanced by ghrelin, while [D-Lys3]-GHRP-6 was only able to partly block this effect and TTX had no effect on muscle strip contraction. It was identified that ghrelin had no effect on the contractive rhythm of the strips. GHS-R1s were located differentially depending on the cell type, including myenteric nerve cells, interstitial cells of Cajal and smooth muscle cells. In conclusion the present study demonstrated that ghrelin may act as an adjuvant to regulate gastric smooth muscle contraction induced by CCh through GHS-R1s, which are expressed on myenteric nerve cells, Cajal cells and smooth muscle cells. Ghrelin may exert its effects by influencing the functional status of different cell types in the gastric muscle layer to subsequently enhance the contractive effect of cholinergic neurotransmitters and enhance gastric motility.