A DNA segment that is highly conserved in glucokinase (hexokinase IV) and hexokinase I cDNA was used to identify specific cDNAs in a library prepared from rat adipose tissue mRNA. Some of these cDNAs were identified as being hexokinase I cDNA. Others, although similar to both the glucokinase and hexokinase I cDNAs, were unique. Two of these unique cDNAs overlapped and contained an open reading frame that encoded a protein of 103 kDa which, when expressed in Escherichia coli, had kinetic properties characteristic of hexokinase II. The entire hexokinase II mRNA sequence and the exon-intron structure of the hexokinase II gene were determined. A single transcription initiation site and two distinct termination sites account for the two observed hexokinase II RNA species of 5500 and 4400 nucleotides that were detected when either of the cDNAs was used as a hybridization probe against poly(A)+ RNA isolated from rat adipose tissue. Hexokinase II mRNA was decreased in adipose tissue from diabetic rats, but was restored by insulin treatment to levels found in nondiabetic control rats. Insulin also induced hexokinase II mRNA in two adipose cell lines (3T3-F442A and BFC-1B) and two skeletal muscle cell lines (C2C12 and L6). In L6 cells, this increase was accounted for by a corresponding increase of hexokinase II gene transcription. Comparison of the structures of the hexokinase II and glucokinase genes support the hypothesis that the 100-kDa hexokinase arose by gene duplication and tandem ligation of a 50-kDa glucokinase-like ancestral gene.