Transforming growth factor-alpha (TGF-alpha) gene transcription is regulated by both epidermal growth factor (EGF) and glucose. Previous studies have suggested that the metabolism of glucose to glucosamine through the enzyme L-glutamine: D-fructose-6-phosphate amidotransferase (GFAT) plays a critical role in the glucose signaling. In this paper, we compared the role of GFAT in the glucose and EGF signals. We found that, although EGF stimulates GFAT mRNA accumulation in MDA-MB-468 cells, this effect of EGF occurred several hours after TGF-alpha transcription increased. MDA-MB-468 cells also exhibited a TGF-alpha transcriptional response to low concentrations of glucose. The TGF-alpha response to glucose but not EGF could be inhibited by a blocker of GFAT activity. Blockade of GFAT was confirmed by using Western blotting with the RL2 antibody, which recognizes an epitope on proteins containing N-acetylglucosamine. Exposure of cells to glucose increased the RL2 signal on several polypeptides, but this change could be blocked by inhibition of GFAT. These results support the notion that glucose stimulation of TGF-alpha expression requires GFAT, but EGF stimulation does not.