The intravenous calcium injection test has been reported to be useful for the diagnosis of gastrinoma. However, the mechanism underlying calcium-evoked gastrin release is not fully understood. We investigated the mechanism of calcium-stimulated gastrin release from gastrinoma cells in vitro with a particular focus on the calcium-sensing receptor (CaR). Human gastrinoma cells were taken from mechanically minced gastrinoma tissues obtained at surgery. In the perifusion system, high [Ca2+]o induced gastrin release from gastrinoma cells. As [Ca2+]o increased, [Ca2+]i rapidly increased, as monitored by fluorometry. The response was not inhibited by nifedipine, a blocker of the voltage-dependent calcium channel. Reverse transcriptase-polymerase chain reaction and subsequent Southern blot hybridization revealed the presence of the CaR gene in human gastrinoma tissues. Moreover, the expression of CaR in gastrinoma tissues was confirmed by immunohistochemistry. Our results demonstrated that CaR was expressed in human gastrinoma cells and could be involved in the mechanism of calcium-evoked gastrin release.