The purpose of the present study was to determine whether human gastric mucous epithelial cells express a functional Ca²⁺-sensing receptor (CaR). Human gastric mucous epithelial cells were isolated from surgical tissues and cultured on glass coverslips, plastic dishes, or porous membrane filters. Cell growth was assessed by the MTT assay, CaR localization was detected by immunohistochemistry and confocal microscopy, CaR protein expression was assessed by Western immunoblotting, and intracellular Ca²⁺ concentration ([Ca²⁺]_i) was determined by fura 2 spectrofluorometry. In paraffin sections of whole stomach, we found strong CaR immunohistochemical staining at the basolateral membrane, with weak CaR-staining at the apical membrane in mucous epithelial cells. Confocal microscopy of human gastric mucous epithelial cell cultures showed abundant CaR immunofluorescence at the basolateral membrane and little to no CaR immunoreactivity at the apical membrane. Western immunoblot detection of CaR protein in cell culture lysates showed two significant immunoreactive bands of 140 and 120 kDa. Addition of extracellular Ca²⁺ to preconfluent cultures of human gastric mucous epithelial cells produced a significant proliferative response. Changes in [Ca²⁺]_iwere also observed in response to graded doses of extracellular Ca²⁺ and Gd³⁺. The phospholipase C inhibitor U-73122 specifically inhibited Gd³⁺-induced changes in [Ca²⁺]_iin the gastric mucous epithelial cell cultures. In conclusion, we have identified the localization of a functional CaR in human gastric mucous epithelial cells.