We confirmed that release of 20-HETE from the isolated perfused kidney of diabetic rats is greatly reduced when compared to age-matched control rats. The present studies were undertaken to examine potential mechanisms for the deficit in renal 20-HETE in rats with streptozotocin-induced diabetes of 3-4 weeks duration. A role for oxidative stress was excluded as treatment of diabetic rats with tempol, a SOD mimetic, for 4 weeks was without effect on the renal release of 20-HETE. Similarly, chronic inhibition of NO formation with L-NAME or aldose reductase with zopolrestat failed to alter the release of 20-HETE from the diabetic rat kidney. As 20-HETE may be metabolized by cyclooxygenase, which expression/activity is increased in diabetes, we included indomethacin in the perfusate of the isolated kidney to inhibit COX but found no effect on 20-HETE release. Fenofibrate treatment of diabetic rats was undertaken for 3 weeks to increase expression of CYP4A in an attempt to find an intervention that would restore release of 20-HETE from the diabetic rat kidney. However, fenofibrate reduced 20-HETE release in both diabetic and control rat kidneys, despite increasing expression of CYP4A. Only insulin treatment of diabetic rats for 2 weeks to reverse the hyperglycemia and maintain blood glucose levels below 200mg/dl reversed the renal deficit in 20-HETE. We conclude that oxidative stress, increased aldose reductase activity or increased COX activity do not contribute to the renal deficit of 20-HETE in diabetes, which may be directly related to insulin deficiency.