Glomerular endothelial cells (GEC) are strategically situated within the capillary loop and adjacent to the glomerular mesangium. GEC serve as targets of metabolic, biochemical and hemodynamic signals that regulate the glomerular microcirculation. Unequivocally, hyperglycemia, hypertension and the local renin angiotensin system partake in the initiation and progression of diabetic nephropathy (DN). Whether free fatty acids (FFA) and reactive oxygen species (ROS) that have been associated with the endothelial dysfunction of diabetic macrovascular disease also contribute to DN is not known. Since endothelial cells from different organs and from different species may display different phenotypes we employed human GEC to investigate the effect of high glucose (22.5 mmol/L), FFA (800 μmol/L) and Angiotensin II (Ang II, 10 ^-7 mol/L) on the genesis of ROS and their effects upon endothelial nitric oxide synthase (eNOS), COX 2, and the synthesis of prostaglandins (PGs). We demonstrated that high glucose but not high FFA increased the expression of a dysfunctional eNOS as well as increased ROS from NADPH oxidase (100%) and likely from uncoupled eNOS. Ang II also induced ROS from NADPH oxidase. High glucose and Ang II up-regulated (100 %) COX 2 via ROS and significantly increased the synthesis of prostacyclin (PGI₂) by 300%. In contrast, FFA did not upregulate COX 2 but increased PGI₂ (500%). These novel studies are the first in human GEC that characterize the differential role of FFA, hyperglycemia and Ang II upon the genesis of ROS, COX2 and prostaglandins and their interplay in the early stages of hyperglcyemia.