We tested the hypothesis that hyperglycemia alters retrograde coronary collateral blood flow by a nitric oxide-mediated mechanism in a canine Ameriod constrictor model of enhanced collateral development. Administration of 15% dextrose to increase blood glucose concentration to 400 or 600 mg/dl decreased retrograde blood flow through the left anterior descending coronary artery to 78 ± 9 and 82 ± 8% of baseline values, respectively. In contrast, saline or l-arginine (400 mg · kg−1 · h−1) had no effect on retrograde flow. Coronary hypoperfusion and 1 h of reperfusion decreased retrograde blood flow similarly in saline- orl-arginine-treated dogs (76 ± 11 and 89 ± 4% of baseline, respectively), but these decreases were more pronounced in hyperglycemic dogs (47 ± 10%). l-Arginine prevented decreases in retrograde coronary collateral blood flow during hyperglycemia (100 ± 5 and 95 ± 6% of baseline at blood glucose concentrations of 400 and 600 mg/dl, respectively) and after coronary hypoperfusion and reperfusion (84 ± 14%). The results suggest that hyperglycemia decreases retrograde coronary collateral blood flow by adversely affecting nitric oxide availability.