The structure of the gene for the Type III isozyme of human hexokinase is nearly identical to that of previously characterized genes for other isozymes of hexokinase. The most striking difference is that the 5'-untranslated sequence and the initial coding sequence are contained in two exons in the Type III hexokinase gene but in a single exon in genes for the other isozymes. Sequence at the transcriptional start site for rat Type III hexokinase (S. Sebastian, J. A. White, and J. E. Wilson, 1999, J. Biol. Chem. 274, 31700-31706) is conserved in the human gene, as is an Oct-1 site, in reverse orientation, approximately 30 bp upstream from the start site. This site has been shown to regulate transcription of both human and rat genes for Type III hexokinase. Comparison of the genes for the various mammalian isozymes of hexokinase indicates that a major feature in the evolution of this isozyme family has been acquisition of alternative first exons.