BACKGROUND/AIMS: Antihypertensive therapies such as angiotensin-converting enzyme-1 inhibitors (ACEi) slow the decline in renal function seen with diabetic nephropathy, although there is still progression ultimately to end-stage renal disease. The aim of this study was to determine if there were added renoprotective benefits seen by combining ACEi with blockade of NADPH oxidase. METHODS: Sprague-Dawley diabetic and non-diabetic rats were randomized to receive intervention therapy with apocynin (15 mg/kg/day, weeks 16-32), apocynin + the ACEi ramipril (1 mg/kg/day, weeks 16-32), or ramipril alone (1 mg/kg). RESULTS: All three treatments retarded the development of albuminuria in the diabetic rats. Apocynin conferred its benefit either as a monotherapy or in combination with ramipril without affecting blood pressure per se. Renal morphological injury was attenuated by all three treatment strategies. Diabetes was associated with increasing renal fibronectin and type IV collagen protein expression, with the combination regimen resulting in the highest decrease in extracellular matrix accumulation. All three treatments prevented the diabetes-associated increases in renal cytosolic superoxide generation as well as urinary isoprostanes. While renal TGF-beta1 activation was reduced by ramipril treatment but not by apocynin as a monotherapy, kidney cortical membranous VEGF was reduced by apocynin as monotherapy and dual therapy but not by ramipril alone. CONCLUSIONS: Combination of NADPH oxidase blockade with ACE inhibitors is a promising regimen which warrants further investigation as a way to confer additional renoprotection in diabetes.