Hyperglycemia-induced alterations in mesangial cell function and extracellular matrix (ECM) protein accumulation are seen in diabetic glomerulopathy. Transforming growth factor-beta1 (TGF-1) mediates high glucose-induced matrix production in the kidney. Recent studies have demonstrated that some of the effects of high glucose on cellular metabolism are mediated by the hexosamine biosynthesis pathway (HBP) in which fructose-6-phosphate is converted to glucosamine 6-phosphate. We have previously shown that the high glucose-mediated fibronectin and laminin synthesis in mesangial cells is mediated by the HBP and that glucosamine is more potent than glucose in inducing TGF-1 promoter luciferase activity. In this study, we investigated the hypothesis that the effects of glucose on mesangial matrix production occur via hexosamine regulation of TGF-1. Culturing SV-40 transformed rat kidney mesangial (MES) cells in 25 mM glucose (HG) for 48 hr increases cellular fibronectin and laminin levels approximately 2-fold on Western blots when compared to low glucose (LG, 5 mM). Glucosamine (1.5 mM, GlcN) or TGF-1 (2.5-5 ng/ml) for 24 - 48 hr also increases ECM synthesis. However, the effects of HG or GlcN with TGF-1 are not additive. The presence of anti-TGF-1 antibodies (20 µg/ml) blocks both TGF-1 and GlcN-induced fibronectin synthesis. TGF-1 increased ECM levels via PKA (laminin and fibronectin) and PKC (fibronectin) pathways. Similarly, TGF-1 and hexosamines led to non-additive increases in phosphorylation of the cyclic AMP Responsive Element (CRE) binding transcription factor (CREB). These results suggest that the effects of excess glucose on mesangial ECM synthesis occur via HBP mediated regulation of TGF-1.