In mammalian tissues, the phosphorylation of intracellular glucose to glucose-6-phosphate (Glu-6-P) is facilitated by four distinct hexokinase (HK) isoenzymes, designated as HKI-IV. Because of the role of HKII as a leading glycolytic enzyme in insulin-sensitive tissues such as skeletal muscle, heart, and adipose tissue, defects in HKII function could contribute to the development of insulin resistance and perhaps Type 2 diabetes. As a first step towards elucidation of the physiological role of HKII in insulin resistance and type 2 diabetes using mouse knock-out models, we determined the genomic structure, sequence of the cDNA and of 4.8 kb of the 5' regulatory region, and tissue-specific expression of the mouse HKII gene. The gene comprises 18 exons that span approximately 50 kb of DNA. Nucleotide sequence of the proximal promoter revealed a number of conserved putative transcription factor binding motifs. We also found numerous repeat elements throughout the mouse HKII gene. The mouse HKII cDNA is approximately 5.5 kb in length and contains an open reading frome of 2751 bp encoding a protein of 917 amino acids. The mouse HKII gene is predominantly expressed in skeletal muscle, heart, and adipose tissue. The transcription initiation and polyadenylation sites for the mouse HKII mRNA were similar to those of the rat and human genes.