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