Increased production of reactive oxygen species (ROS) in diabetes may be a common pathway linking diverse pathogenic mechanisms of diabetic vascular complications, including nephropathy. Assessment of the oxidative stress production pathway is therefore important for the prediction and prevention of diabetic complications. However, ROS production mechanisms remain unclear in diabetic glomeruli. To identify the source and determine the mechanisms of ROS production in the diabetic kidney, diabetes was induced with streptozotocin in rats. After 6 wk, glomerular ROS production had increased in the streptozotocin rat kidney, as assessed by dihydroethidium-derived chemiluminescence. ROS production was increased by the addition of NADH or l-arginine and was partially reduced by the addition of diphenylene iodonium or N^G-nitro-l-arginine methyl ester, identifying NAD(P)H oxidase and nitric oxide (NO) synthase (NOS) as ROS sources. The mRNA and protein expression of endothelial NOS (eNOS), as measured by real-time RT-PCR and Western blotting, increased significantly (mRNA level, 1.3-fold; protein level, 1.8-fold). However, the dimeric form of eNOS was decreased in diabetic glomeruli, as measured by low-temperature SDS-PAGE. Production of renal ROS and NO by uncoupled NOS was imaged by confocal laser microscopy after renal perfusion of 2′,7′-dichlorofluorescein diacetate (a ROS marker) and diaminorhodamine-4M AM (a NO marker) with l-arginine. Accelerated ROS production and diminished bioavailable NO caused by NOS uncoupling were noted in the diabetic kidney. Administration of tetrahydrobiopterin (BH₄), a cofactor for eNOS, reversed the decreased dimeric form of eNOS and glomerular NO production. Our results indicate that NAD(P)H oxidase and uncoupling of eNOS contribute to glomerular ROS production, mediated by the loss of BH₄ availability. These mechanisms are potential key targets for therapeutic interventions.