Reports differ as to the efficacy of glucose and insulin as cardioplegic additives. Although deliberate oxygenation of crystalloid cardioplegic solutions improves myocardial protection, little is known about the protection afforded by glucose and insulin in such oxygenated solutions. In the isolated working rat heart, we studied the addition of oxygen, glucose, and insulin, separately and together, to a cardioplegic solution. The solution was equilibrated with O2 or N2, with glucose added as a substrate or sucrose as a nonmetabolizable osmotic control, with or without insulin. Hearts were arrested for 2 hours at 8 °C by multidose infusions. Oxygenation decreased lactate production and improved high-energy phosphate and glycogen preservation during arrest, prevented ischemic contracture, and improved functional recovery. The addition of glucose to the oxygenated solution increased the level of adenosine triphosphate at end-arrest from 10.5 ± 0.5 to 13.9 ± 0.6 nmol/mg dry weight and glycogen stores from 18.7 ± 2.5 to 35.7 ± 5.5 nmol/mg dry weight. The further addition of insulin did not better preserve these metabolites. Improvements in functional recovery due to glucose or insulin in the oxygenated solution attained statistical significance when both additives were included. Glucose increased lactate production significantly only when the solution was nitrogenated. Insulin added to the nitrogenated glucose-containing solution increased adenosine triphosphate and glycogen levels after 1 hour of arrest; and, although insulin did not prevent ischemic contracture from developing during the latter part of arrest with profound depletion of these metabolites, functional recovery was improved. The mechanism of improved functional recovery by insulin is not clear. Whether aerobic or anaerobic metabolism is favored during arrest modifies the effects of glucose and insulin. The data support the use of a fully oxygenated cardioplegic solution containing glucose, and perhaps insulin, for myocardial protection, and suggest that these additives may be beneficial whether the cardioplegic solution is deliberately oxygenated or not.