Myocardial levels of ammonia, glutamate, and glutamine and the release of glutamate and glutamine were studied in the isolated perfused rat heart during perfusion with ammonium chloride, epinephrine, and conditions of anoxia or ischaemia. Perfusion for 15 min with effective ammonium chloride concentrations of 0.53, 0.71, and 2.06 mmol/l resulted in glutamine production of 1.34, 0.95, and 4.41 mmol·15 min−1/200 mg dry weight compatible with the presence of glutamine synthetase in rat myocardium. Myocardial ammonium content was unchanged by perfusion with 0.53 and 0.71 mmol/l ammonium chloride, but was increased by 1.36 μmol·15 min−1/200 mg dry weight by perfusion with 2.06 mmol/l ammonium chloride. Increased myocardial contents of ammonia and glutamine were not accompanied by depression of left ventricular pressure. Perfusion with epinephrine (0.20 μg/ml) resulted in an increased myocardial content of glutamine. Anoxia or ischaemia resulted in no changes in ammonia content, and no changes in glutamine or glutamate production. The net release of glutamine into the perfusate was about 10 times the net release of glutamate.