We identified two enhancer elements of the mouse GLUT1 gene responsive to serum, growth factor, and oncogenes; the first enhancer element (enhancer-1) is located 2.7 kilobases upstream of the cap site of the gene, and the second one (enhancer-2) is located in the second intron of the gene (Murakami, T., Nishiyama, T., Shirotani, T., Shinohara, Y., Kan, M., Ishii, K., Kanai, F., Nakazuru, S., and Ebina, Y. (1992) J. Biol. Chem. 267, 9300-9306). In the present work, we describe the role of insulin in activation of these two enhancers. NIH/3T3 HIR3.5 cells, which express a large number of insulin receptors, were stably transformed by hybrid genes containing the enhancer(s) and promoter of GLUT1 gene and the coding region of chloramphenicol acetyltransferase (CAT) gene as a reporter gene. In stable transformants of the reporter gene without the enhancers, the CAT mRNA was not induced by insulin; however, in clones containing the reporter gene with enhancer-1, the CAT mRNA was induced by insulin at 30 min and reached a maximum at 1 h. In clones transfected by the reporter gene with enhancer-2, the CAT mRNA was induced at 1 h and reached a maximum at 3 h. To determine the early response element to insulin in enhancer-1, transformants of hybrid reporter genes containing truncated or mutated enhancer-1 were examined. The homologous sequence with the serum response element in enhancer-1 is essential for an early response to insulin.