Ghrelin is an endogenous ligand for the growth hormone secretagogue receptor. The presence of ghrelin in pancreatic islet cells has been previously reported and it is known to increase the [Ca2+]i in (-cells, affecting insulin secretion. However, evidence for the existence of the ghrelin system and its calcium signalling pathway in the exocrine pancreas remains unclear. Thus this study aims, first, to investigate the expression of ghrelin and its receptor in pancreatic AR42J cells and, secondly, to elucidate its calcium signalling pathway. Our results showed that ghrelin and ghrelin receptor were consistently expressed in AR42J cells. Moreover, fluorescence imaging showed that cholecystokinin-8, ghrelin and growth hormone-releasing hexapeptide stimulate [Ca2+]i in AR42J cells in a dose-dependent manner. Ghrelin and the hexapeptide produced a biphasic elevation in [Ca2+]i with an initial transient increase, followed by a sustained plateau. In the presence of (D-Lys3)-GHRP-6, the [Ca2+]i evoked by ghrelin was suppressed. In the absence of extracellular Ca2+, the transient phase of the ghrelin response was maintained but greatly diminished while the plateau phase was completely abolished. Pretreatment with 2-aminoethoxydiphenyl borate and xestospongin C abolished the transient phase and inhibited the sustained phase of the ghrelin response. The stimulatory effect of ghrelin was also blocked by nifedipine. These results indicate that ligand stimulation of the ghrelin receptor could lead to a biphasic [Ca2+]i mobilization in these cells. These data suggests the presence of a ghrelin system in pancreatic AR42J cells. In addition, its roles in exocrine function are implicated in the pancreas.