Vascular endothelial growth factor (VEGF) is implicated in the development of proteinuria in diabetic nephropathy. High ambient glucose present in diabetes stimulates VEGF expression in several cell types, but the molecular mechanisms are incompletely understood. Here, primary culture rat mesangial cells served as a model to investigate the signal transduction pathways involved in high glucose-induced VEGF expression. Exposure to high glucose 25mM significantly increased VEGF mRNA evaluated by real-time PCR by 3h, VEGF cellular protein content assessed by immunoblotting or immunofluorescence within 24 h, and VEGF secretion by 24 h. High glucose-induced VEGF expression was blocked by an antioxidant, Tempol, and antisense oligonucleotides directed against p22^phox, a NADPH oxidase subunit. Inhibition of protein kinase C (PKC)-₁ with the specific pharmacological inhibitor LY333531 or inhibition of PKC- with a cell permeable specific pseudosubstrate peptide also prevented enhanced VEGF expression in high glucose. Enhanced VEGF secretion in high glucose was prevented by Tempol, PKC-₁, or PKC- inhibition. In normal glucose 5.6mM, over expression of p22^phox or constitutively active PKC- enhanced VEGF expression. Hypoxia inducible factor-1 protein was significantly increased in high glucose only by 24h, suggesting a possible contribution to high glucose-stimulated VEGF expression at later time points. Thus, reactive oxygen species generated by NADPH oxidase, and both PKC-₁ and -, play important roles in high glucose-stimulated VEGF expression and secretion by mesangial cells.