In mammalian tissues hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) exists as four isoenzymes encoded by distinct genes. These proteins are homologous and are organized in two homologous domains, with the exception of hexokinase type IV which has only one. This organization is believed to be the result of a duplication and tandem fusion event involving the gene encoding for the ancestral hexokinase. In this study, we cloned the carboxyl-domain of human hexokinase type III and expressed it in Escherichia coli as a glutathione S-transferase fusion protein, using the pGEX-2T expression vector. The recombinant protein showed catalytic activity. A comparative study of the kinetic properties of the expressed carboxyl-domain and the enzyme partially purified from human lymphocytes is also shown. The results now allow a better understanding of the role of the carboxyl-domain in determining the catalytic properties of the enzyme.