Combination therapy of ACE inhibition and AT1 receptor blockade has been shown to provide greater renoprotection than ACE inhibitor alone in human diabetic nephropathy, suggesting that ACE-independent pathways for ANGII formation are of major significance in disease progression. Studies were performed to determine the magnitude of intrarenal ACE-independent formation of ANGII in type II diabetes. Although renal cortical ACE protein activity (2.1 ± 0.8 vs 9.2 ± 2.1 AFU/mg/min) and intensity of immunohistochemical staining were significantly reduced and ACE2 protein activity (16.7 ± 3.2 vs 7.2 ± 2.4 AFU/mg/min) and intensity elevated, kidney ANGI (113 ± 24 vs 110 ± 45 fmol/g) and ANGII (1, 017 ± 165 vs 788 ± 99 fmol/g) levels were not different between diabetic and control mice. Afferent arteriole vasoconstriction due to conversion of ANGI to ANGII was similar in magnitude in kidneys of diabetic (-28 ± 3% at 1 μM) and control (-23 ± 3% at 1 μM) mice; a response completely inhibited by AT₁ receptor blockade. In control kidneys, afferent arteriole vasoconstriction produced by ANGI was significantly attenuated by ACE inhibition, but not by serine protease inhibition. In contrast, afferent arteriole vasoconstriction produced by intrarenal conversion of ANGI to ANGII was significantly attenuated by serine protease inhibition, but not by ACE inhibition in diabetic kidneys. In conclusion, there is a switch from ACE-dependent to serine protease-dependent ANGII formation in the type II diabetic kidney. Pharmacological targeting of these serine protease-dependent pathways may provide further protection from diabetic renal vascular disease.