Using the perfused kidneys of age-matched controls and streptozotocin (STZ)-induced diabetic rats, we previously demonstrated that endothelial dysfunction is present in STZ-induced diabetic rats and that acetylcholine (ACh) increases the level of 6-keto-prostaglandin F_1α (a metabolite of prostacyclin) in the effluent from such perfused kidneys. Here, we investigated whether the ACh-induced relaxation in the perfused kidney is modulated by prostacyclin and/or thromboxane A₂ (TXA₂) in the STZ-induced diabetic state. ACh-induced renal vasodilatation was significantly weaker in STZ-induced diabetic rats than in age-matched controls, and it was not affected by treatment with 10 μM furegrelate (TXA₂-synthase inhibitor) or 1 μM SQ29548 (TXA₂- receptor antagonist) in either group. However, it was attenuated by 10 μM tranylcypromine (prostacyclin-synthesis inhibitor), but only in the diabetic group. These results suggest that the endothelium-dependent relaxation induced by ACh in the renal vascular bed of STZ-induced diabetic rats is regulated by prostacyclin, not by TXA₂. Increased prostacyclin-signaling may occur to help compensate for the impaired endothelial function seen in the kidney in long-term diabetic states.