Prolonged treatment of 3T3-L1 adipocytes decreases expression of GLUT4, the insulin-responsive glucose transporter. Expression of promoter-reporter gene constructs that contained 2900 or 785 base pairs of 5'-flanking region of the murine GLUT4 gene was down-regulated by insulin (p < 0.0005), whereas expression of constructs that contained 641, 469, or 78 base pairs of 5'-flanking region was not. Nuclear extract from 3T3-L1 adipocytes protected the region from -707 to -681 in the GLUT4 5'-flanking region from DNase I digestion. Using an oligonucleotide probe that corresponded to this footprinted region, two major protein-DNA complexes were identified by a gel mobility shift assay. Southwestern analysis identified four protein bands with molecular masses from 38 to 46 kDa that bound to the insulin-responsive region probe. A reporter gene construct in which bases -706 to -676 were deleted was not repressed by insulin treatment, confirming that this sequence is necessary for the repression of the GLUT4 promoter by insulin in 3T3-L1 adipocytes. This sequence does not show homology to previously described insulin response elements and thus represents a distinct mechanism of gene regulation by insulin.