The type I, II, and III isozymes of mammalian hexokinase (100 kDa) all consist of a duplicated, highly homologous peptide sequence, each half of which is very similar to that of glucokinase (type IV hexokinase, 50 kDa) and yeast hexokinase (50 kDa). We isolated a genomic clone of type II hexokinase that contained five exons encoding the C-terminal region of type II hexokinase. The positions of intron insertions of the isolated clone were found to be identical with those of the glucokinase gene, indicating that the type II hexokinase gene arose from the glucokinase gene. Furthermore, we prepared two DNA fragments of the type II hexokinase gene amplified from total genomic DNA. The exons in these fragments were found to be constructed by linkage of the coding region of the last exon and second exon of the glucokinase gene, indicating that the hexokinase gene arose by fusion of two glucokinase genes. These results clearly show that the mammalian hexokinase gene evolved from the glucokinase gene by gene duplication and fusion with conservation of the gene organization.