The mammalian hexokinase (HK) family includes three closely related 100-kDa isoforms (HKI-III) that are thought to have arisen from a common 50-kDa precursor by gene duplication and tandem ligation. Previous studies of HKI indicated that a glucose 6-phosphate (Glu-6-P)-regulated catalytic site resides in the COOH-terminal half of the molecule and that the NH2-terminal half contains only a Glu-6-P binding site. In contrast, we now show that proteins representing both halves of human and rat HKII have catalytic activity and that each is inhibited by Glu-6-P. The intact enzyme and the NH2- and COOH-terminal halves of the enzyme each increase glucose utilization when expressed in Xenopus oocytes. Mutations corresponding to either Asp-209 or Asp-657 in the intact enzyme completely inactivate the NH2- and COOH-terminal half enzymes, respectively. Mutation of either of these sites results in a 50% reduction of activity in the 100-kDa enzyme. Mutation of both sites results in a complete loss of activity. This suggests that each half of the HKII molecule retains catalytic activity within the 100-kDa protein. These observations indicate that HKI and HKII are functionally distinct and have evolved differently.