When cold storage techniques used in cardiac transplantation are extended beyond 3 hours, there is significant depression in ventricular function. This study was undertaken to determine whether the addition of the amino acid L-glutamate or the oxygen free-radical scavengers superoxide dismutase (SOD) and catalase (CAT) during extended periods of cold storage would improve ventricular function. Fifteen rabbit hearts were placed on a Langendorff apparatus, arrested with crystalloid potassium cardioplegia, stored in iced saline solution (3 degrees C) for 5 hours, and then reperfused at 37 degrees C for 1 hour. In five hearts L-glutamate (4 mmol/L) was added to the cardioplegic and reperfusate solutions, and five hearts received SOD (1500 units/kg/L) and CAT (3500 units/kg/L), whereas in five others the cardioplegic and reperfusate solutions were unmodified. Hearts treated with L-glutamate had the best recovery of positive dP/dt (79%* glutamate vs 49%* SOD and CAT vs 36% unmodified), negative dP/dt (76%* glutamate vs 53% SOD and CAT vs 45% unmodified), developed pressure (67%* glutamate vs 51% SOD and CAT vs 45% unmodified), and coronary flow (81%* glutamate vs 79%* SOD and CAT vs 62% unmodified). We conclude that substrate enhancement with L-glutamate provides superior myocardial protection than is possible with the oxygen free-radical scavengers SOD and CAT during extended periods of cold storage for cardiac transplantation.