γ-Glutamyl transpeptidase possesses two histidine residues at positions 383 and 505 which are conserved in all mammalian and bacterial species. In order to elucidate the functions of these residues, we prepared mutants in which these residues were replaced by Ala. Kinetic analysis of the hydrolysis of L-γ-glutamyl-p-nitroanilide indicated that substitution at His-383 decreased the V_max value to 14% of that of the wild type, but had no effect on V_max/K_m. In reactions involving glycylglycine as the acceptor substrate, the V_max value of this mutant decreased to 38% with little alteration of V_max/K_m for L-γ-glutamyl-p-nitroanilide as a γ-glutamyl donor, but with a significant reduction of V_max/K_m for the acceptor. These results show that this substitution causes impairment of the step in which the free enzyme is regenerated from the γ-glutamyl enzyme by water or an acceptor substrate. On the other hand, replacement of His-505 resulted in a decrease of the V_max value for transpeptidation to about 10% of that of the wild type despite no substantial effect on the V_max value for the hydrolysis reaction. However, this substitution did not affect V_max/K_m for the acceptor on transpeptidation. Thus, the formation of a non-productive enzyme-substrate complex with the acceptor substrate would decrease the V_max value on transpeptidation. These results suggest that His-383 plays an important catalytic role in facilitating the degradation of the γ-glutamyl-enzyme through hydrolysis or transfer of the γ-glutamyl moiety to an acceptor. It was also shown that His-505 is important in the formation of a complex of the γma;-glutamyl enzyme with the acceptor substrate even though it plays no critical role in the catalysis. Although the pH-dependence profile and the van't Hoff plot for the ionic group responsible for enzyme activity were consistent with the requirement of a histidine residue, neither of the conserved histidines could be assigned as such an ionic group. This suggests that another histidine residue (s) might play an essential role in the enzyme function.