The calcium-sensing receptor (CaR) is a G protein-coupled receptor. The CaR stimulation elicits phospholipase C-mediated inositol triphosphate formation, leading to an elevation in the level of intracellular calcium released from endoplasmic reticulum (ER). Depletion of ER Ca(2+) leads to ER stress, which is thought to induce apoptosis. Intracellular calcium overload-induced apoptosis in cardiac myocytes during hypoxia-reoxygenation (H/Re) has been demonstrated. However, the links between CaR, ER stress and apoptosis during H/Re are unclear. This study hypothesized that the CaR could induce apoptosis in neonatal rat cardiomyocytes during H/Re via the ER stress pathway. Neonatal rat cardiomyocytes were subjected to 3 hr of hypoxia, followed by 6 hr of reoxygenation. CaR expression was elevated and the number of apoptotic cells was significantly increased, as shown by transferase-mediated dUTP nick end-labelling, with exposure to CaCl(2), a CaR activator, during H/Re. The intracellular calcium concentration was significantly elevated and the Ca(2+) concentration in the ER was dramatically decreased during H/Re with CaCl(2); both intracellular and ER calcium concentrations were detected by laser confocal microscopy. Expression of GRP78 (glucose-regulated protein 78), the cleavage products of ATF6 (activating transcription factor 6), phospho-PERK [pancreatic ER kinase (PKR)-like ER kinase], the activated fragments of caspase-12, and phospho-JNK (c-Jun NH(2)-terminal kinase) were increased following exposure to CaCl(2) during H/Re. Our results confirmed that the activated CaR can induce cardiomyocyte apoptosis via ER stress-associated apoptotic pathways during H/Re.