Increased extracellular Ca(2+) ([Ca(2+)](o)) can damage tissues, but the molecular mechanisms by which this occurs are poorly defined. Using HEK 293 cell lines that stably overexpress the Ca(2+)-sensing receptor (CaR), a G protein-coupled receptor, we demonstrate that activation of the CaR leads to apoptosis, which was determined by nuclear condensation, DNA fragmentation, caspase-3 activation, and increased cytosolic cytochrome c. This CaR-induced apoptotic pathway is initiated by CaR-induced accumulation of ceramide which plays an important role in inducing cell death signals by distinct G protein-independent signaling pathways. Pretreatment of wild-type CaR-expressing cells with pertussis toxin inhibited CaR-induced [(3)H]ceramide formation, c-Jun phosphorylation, and caspase-3 activation. The ceramide accumulation, c-Jun phosphorylation, and caspase-3 activation by the CaR can be abolished by sphingomyelinase and ceramide synthase inhibitors in different time frames. Cells that express a nonfunctional mutant CaR that were exposed to the same levels of [Ca(2+)](o) showed no evidence of activation of the apoptotic pathway. In conclusion, we report the involvement of the CaR in stimulating programmed cell death via a pathway involving GTP binding protein alpha subunit (Galpha(i))-dependent ceramide accumulation, activation of stress-activated protein kinase/c-Jun N-terminal kinase, c-Jun phosphorylation, caspase-3 activation, and DNA cleavage.