Post-ischemic reperfusion may precipitate cardiomyocyte death upon correction of intracellular acidosis due in part to mitochondrial permeability transition. We investigated whether glycine, an amino acid with poorly understood cytoprotective properties, may interfere with this mechanism. In cardiomyocyte cultures, addition of glycine during re-energization following 1h of simulated ischemia (NaCN/2-deoxyglucose, pH 6.4) completely prevented necrotic cell death associated to pH normalization. Glycine also proteccted against cell death associated to pH normalization in reoxygenated rat hearts. Glycine prevented cyclosporin-sensitive swelling and calcein release associated to re-energization in rat heart mitochondria submitted to simulated ischemia or to Ca2+ stress under normoxia. NMR-spectroscopy revealed a marked glycine depletion in re-energized cardiomyocytes that was reversed by exposure to 3mM glycine.. These results suggest that intracellular glycine exerts a previously unrecognized inhibition on mitochondrial permeability transition in cardiac myocytes, and that intracellular glycine depletion during myocardial hypoxia/reoxygenation makes the cell more vulnerable to necrotic death.