We previously observed elevated plasma levels of citrulline and arginine in diabetic retinopathy patients compared to diabetic controls. We tested our hypothesis that citrulline plus arginine induces angiogenesis and increases permeability in retinal endothelial cells. Human retinal microvascular endothelial cells (HRMECs) were treated with citrulline, arginine, or citrulline + arginine, and angiogenesis was measured with cell proliferation, migration, and tube formation assays. Permeability was measured in HRMEC monolayers via trans-endothelial electrical resistance (TEER) and FITC-labeled dextran. We also measured arginase activity, arginase-1 and arginase-2 expression, protein expression and phosphorylation of endothelial nitric oxide synthase (eNOS), and nitric oxide (NO) production. Citrulline + arginine induced endothelial cell proliferation (