Increase in free intracellular calcium [Ca 2+]i plays a crucial role in cardiomyocyte ischemic injury. Here we demonstrate that overexpression of the sarcoplasmic-reticulum stress-protein Grp94 reduces myocyte necrosis due to calcium overload or simulated ischemia. Selective three- to eightfold Grp94 increase, with no change in Grp78 or calreticulin amount, was achieved by stable transfection of skeletal C2C12 and cardiac H9c2 muscle cells. After exposure to the calcium ionophore A23187, LDH release from five different Grp94-overexpressing clones of either C2C12 and H9c2 origin was significantly lower than that of control ones and [Ca 2+]i increase was significantly delayed. The number of necrotic cells, evaluated by propidium iodide uptake, was reduced when cells from the Grp94-overexpressing H9c2 clone were exposed to conditions simulating ischemia. Experiments performed in neonatal rat cardiomyocytes co-transfected with grp94 and the green fluorescent protein (GFP) cDNAs validated the protective effect of Grp94 overexpression. A lower percentage of propidium-iodide positive/GFP-fluorescent myocytes co-expressing exogenous Grp94, with respect to myocytes expressing GFP alone, was observed after exposure to either A23187 (6.6% vs. 14.0%, respectively) or simulated ischemia (8.5% vs. 17.7%, respectively). In conclusion, the selective increase in Grp94 protects cardiomyocytes from both ischemia and calcium overload counteracting [Ca 2+]i elevations.