We assessed the effects of 4 weeks of streptozocin-induced diabetes on regional myocardial glycolytic metabolism during ischemia in anesthetized open-chest domestic swine. Diabetic animals were hyperglycemic (12.0 ± 2.1 v 6.6 ± .5 mmol/L), and had lower fasting insulin levels (27 ± 8 v 79 ± 19 pmol/L). Myocardial glycolytic metabolism was studied with coronary flow controlled by an extracorporeal perfusion circuit. Left anterior descending coronary artery (LAD) flow was decreased by 50% for 45 minutes and left circumflex (CFX) flow was constant. Myocardial glucose uptake and extraction were measured with d-[6-3H]-2-deoxyglucose (DG) and myocardial blood flow was measured with microspheres. The rate of glucose conversion to lactate and lactate uptake and output were assessed with a continuous infusion of [6-14C]glucose and [U-13C]lactate into the coronary perfusion circuit. Both diabetic and nondiabetic animals had sharp decreases in subendocardial blood flow during ischemia (from 1.21 ± .10 to 0.43 ± .08 mL · g-1 · min-1 in the nondiabetic group, and from 1.30 ± .15 to 0.55 ± .11 in the diabetic group). Diabetes had no significant effect on myocardial glucose uptake or glucose conversion to lactate under either well-perfused or ischemic conditions. Forty-five minutes of ischemia resulted in significant glycogen depletion in the subendocardium in both nondiabetic and diabetic animals, with no differences between the two groups. Glycolytic metabolism is not impaired in hyperglycemic diabetic swine after 1 month of the disease when compared with that in normoglycemic nondiabetic animals. The myocardial content of the insulin-regulatable glucose transporter (GLUT 4) was measured in left ventricular biopsies. Diabetes resulted in significantly lower GLUT 4 levels in both the subepicardial and subendocardial layers (23% [P < .01] and 11% [P < .05], respectively). There were no subepicardial/subendocardial differences in GLUT 4 levels in either group. In conclusion, 1 month of streptozocin diabetes resulted in a modest decrease in myocardial GLUT 4 levels, but did not affect the rate of glycolysis during myocardial ischemia.