Conversion of normally quiescent mesangial cells into extracellular matrix over-producing myofibroblasts in response to high ambient glucose and transforming growth factor (TFG)-₁ is central to the pathogenesis of diabetic nephropathy. Previously, we reported that mesangial cells respond to high glucose by generating reactive oxygen species (ROS) from NADPH oxidase dependent on protein kinase C (PKC) - activation. We investigated the role of TGF-₁ in this action of high glucose on primary rat mesangial cells within 1 to 48h. Both high glucose and exogenous TGF-₁ stimulated PKC- kinase activity, as measured by an immune complex kinase assay and immunofluorescence confocal cellular imaging. In high glucose, Akt Ser473 phosphorylation appeared within 1 h and Smad2/3 nuclear translocation was prevented with neutralizing TGF-₁ antibodies. Neutralizing TGF-₁ antibodies, or a TGF- receptor kinase inhibitor (LY364947), or a PI3 kinase inhibitor (wortmannin) prevented PKC- activation by high glucose. TGF-₁ also stimulated cellular membrane translocation of PKC-, -₁, -, and -, similar to high glucose. High glucose and TGF-₁ enhanced ROS generation by mesangial cell NADPH oxidase, as detected by 2,7dichlorofluorescein immunofluorescence. This response was abrogated by neutralizing TGF-₁ antibodies, LY364947, or a specific PKC- pseudosubstrate peptide inhibitor. Expression of constitutively active PKC- in normal glucose caused upregulation of p22phox , a likely mechanism of NADPH oxidase activation. We conclude that very early responses of mesangial cells to high glucose include autocine TGF-₁ stimulation of PKC isozymes including PI3 kinase activation of PKC- and consequent generation of ROS by NADPH oxidase.