We have recently shown that glucose and glucosamine regulate the transcription of transforming growth factor-α (TGFα) in rat aortic smooth muscle (RASM) cells. Based on the increased potency of glucosamine compared to glucose, we hypothesized that stimulation of TGFα transcription by glucose is mediated through the hexosamine biosynthesis pathway. The yeast cDNA for the rate-limiting enzyme of this pathway, glutamine:fructose-6- phosphate amidotransferase (GFA), was therefore expressed in RASM cells. GFA- transfected cells showed an increase in GFA activity, exhibiting a 2.2-fold increase in the synthesis of glucosamine-6-phosphate, the first product of the hexosamine biosynthetic pathway. To test the effect of GFA overexpression on TGFα transcriptional activity, cells were transiently cotransfected with GFA along with a reporter plasmid containing the firefly luciferase gene under control of the TGFα promoter. GFA-transfected cells exhibited a glucose-dependent 2-fold increase in TGFα activity compared to control cells. Maximal stimulation of TGFα-luciferase activity by glucosamine, however, was equivalent in GFA- and control-transfected cells, confirming that the stimulation observed by both agents operated through the same pathway. This increase in TGFα activity was inhibited (85% at 0.5 mM glucose and 69% at 30 mM glucose) by the glutamine analog and inhibitor of GFA, 6- diazo-5-oxonorleucine (10 μM). Control studies confirmed that the increased TGFα-luciferase activity in the GFA-expressing cells was not an artifact of altered growth, survival, or transfection efficiency. Experiments using pharmacological agents to stimulate or inhibit protein kinase C and cAMP- dependent kinase do not support a role for these second messengers in the signaling pathway. Tunicamycin inhibited the ability of glucose to stimulate TGFα activity, suggesting that protein glycosylation does play a role. We conclude that products of the hexosamine biosynthesis pathway mediate the stimulation by glucose of TGFα in aortic smooth muscle cells.