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. [Copyright &y& Elsevier]