Ablation of GLUT4 in adipose tissues results in whole body insulin resistance and high-fat feeding down-regulates GLUT4 mRNA in white adipose tissues. Previous studies demonstrated that adipose tissue specific element(s) (ASE) of the murine GLUT4 gene is located between -551 and -442 relative to transcription start site and that high-fat responsive element(s) (HFRE) for down-regulation of the GLUT4 gene is located between bases -1001 and -442. To further characterize these regulatory elements, the regulation of GLUT4 minigenes containing -701, -551, and -506 bp of 5(')-flanking region was studied in transgenic mice. GLUT4 minigene mRNA from -506 transgenic mice did not express in adipose tissues, indicating that ASE located within 45 bp is located between bases -551 and -506. An 80-kDa of nuclear DNA binding protein was found to bind to a -TCCTCGTGGGAAGCG- element located between bases -551 and -537. High-fat diet feeding down-regulated GLUT4 minigene mRNA in -701 transgenic mice, but not in -551 transgenic mice, indicating that HFRE is located within 150 bp between bases -701 and -551 of the GLUT4 gene and is distinct from ASE.