Previous studies from our lab have demonstrated cardiovascular abnormalities such as depressed mean arterial blood pressure and heart rate, endothelial dysfunction and attenuated pressor responses to vasoactive agents in streptozotocin diabetic rats. We investigated whether these abnormalities are due to diabetes-associated chronic activation of inducible nitric oxide synthase (iNOS). Control and streptozotocin (60 mg/kg, iv) diabetic rats were treated with either vehicle or N6-(1-Iminoethyl)-l-lysine dihydrochloride (l-NIL, 3 mg/kg/day, p.o), a specific inhibitor of iNOS for 8 weeks. At the end of treatment, the mean arterial blood pressure and heart rate were measured in freely moving conscious rats. Further, pressor responses to bolus doses of methoxamine were determined. Endothelial nitric oxide synthase (eNOS) and iNOS expression as well as nitrotyrosine (NT) levels were assessed in the heart and superior mesenteric arteries by western blot and immunohistochemistry. Untreated diabetic rats showed depressed mean arterial blood pressure and heart rate and exhibited vascular hyporeactivity that were significantly improved by treatment with l-NIL. Further, decreased eNOS expression and increased iNOS expression and activity were associated with increased NT levels in the heart and superior mesenteric arteries of untreated diabetic rats. l-NIL treatment of diabetic rats normalized the expression of eNOS and NT levels without any effect on iNOS expression in the heart and superior mesenteric arteries. The results of our study suggest that induction of iNOS in cardiovascular tissues contributes significantly to the depressed mean arterial blood pressure, heart rate and pressor responses to vasoactive agents. Chronic inhibition of iNOS in diabetes may prove beneficial in the treatment of cardiovascular abnormalities.