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The amino acid glutamate is used in cardioplegic solutions, yet evidence is conflicting as to whether or not exogenous glutamate is indeed cardioprotective. This controversy may be because increasing extracellular glutamate does not necessarily lead to an increase in intracellular glutamate. In this study we aimed to determine whether isolation of myocytes in the presence of glutamate resulted in glutamate-loading of the cells, and, if so, whether such loading protected myocytes from simulated (chemical) hypoxia. Single ventricular myocytes were isolated from rat hearts in the presence and absence of 6.4 mm glutamate. Levels of glutamate and ATP were determined using HPLC, and NADH/NAD⁺was determined from cell autofluorescence. Chemical hypoxia was induced by superfusion with a solution containing 2.5 m m cyanide and no glucose. Intracellular [Ca²⁺] was measured by loading cells with indo-1 …