We recently identified cysteine-135 as an important amino acid for heparinase I (EC 4.2.2.7) activity. In this study, we have identified a second residue critical for enzymatic activity. We observe concentration-dependent inactivation of heparinase I in the presence of reversible histidine-modifying diethyl pyrocarbonate (DEPC); 0.3 mM DEPC results in 95% of heparinase I inactivation in less than 3 min, and as low as 10 microM DEPC results in a 85% loss of heparinase I activity in 15 min. Heparinase I activity is restored following hydroxylamine treatment. This, along with other experiments, strongly suggests that the inactivation of heparinase I by DEPC is specific for histidine residues. Chemical modification, under nondenaturing conditions, of the histidines using nonradiolabeled and [14C]DEPC indicates that between one and two histidine residues are modified. Chemical modification of the surface-accessible histidines, in the presence and absence of heparin, suggests that the histidine(s) lie(s) in or near the active site of heparinase I. The wild-type heparinase I has four histidine residues; site-directed mutagenesis of H129A, H165A, and H339A did not affect enzyme activity and the kinetic parameters, suggesting that these residues are not essential for heparinase I activity. However, H203A inactivates heparinase I while a H203D mutant has residual activity, indicating a role of this residue in catalysis. We propose that histidine-203, contained in the heparin binding site, is immediately adjacent to cysteine-135, and these residues together form the catalytic domain of heparinase I.