An aspartase purified from Hafnia alvei was inactivated by diethylpyrocarbonate (DEP) in a pseudo-first-order inactivation. The first-order plot was biphasic. The inactivation process was not saturable and the second order rate constant was 1.3 M^(-1)s^(-1). The inactivated aspartase was reactivated with NH₂OH. The difference absorption spectrum of DEP-inactivated vs native enzyme preparations revealed a marked peak around 242 nm. The pH dependence of the inactivation rate suggests that an amino acid residue having a pK value of 7.2 was involved in the inactivation. L-aspartate, fumarate (substrates), and chloride ion (inhibitor) protected the enzyme against inactivation, indicating that histidine residues for the enzyme activity are located at the active site of this aspartase. Inspection of the spectral change at 240 nm along with inactivation in the presence and absence of Cl^- ion demonstrated that the number of essential histidine residues is less than two. Thus, one or two histidines are in or near the aspartate binding site and participate in an essential step of the catalytic reaction.