The potential photoaffinity probe 8-azido-adenosine (8-N3-Ado) was shown to serve as a substrate for the 3'-oxidative activity of human S-adenosylhomocysteine (AdoHcy) hydrolase (Aiyar, V. N., and Hershfield, M. S. (1985) Biochem. J. 232, 643-650). In this study, we have determined the equilibrium binding properties of 8-N3-Ado with AdoHcy hydrolase (NAD+ form) and identified the specific amino acid residues that are covalently modified. After irradiation of the reaction mixture of [2-3H]8-N3-Ado and AdoHcy hydrolase (NAD+ form) and followed by tryptic digestion, peptides specifically photolabeled by [2-3H]3'-keto-8-N3-Ado were effectively separated from peptides nonspecifically labeled with [2-3H]8-N3-Ado using boronate affinity chromatography. After purification by reverse phase high performance liquid chromatography, two photolabeled peptides were isolated and identified as Val175-Lys186 and Val319-Arg327, in which Ala177 and Ile321 were associated with radioactivity. The specificity of the photoaffinity labeling with [2-3H]3'-keto-8-N3-Ado was demonstrated by the observation that these photolabeled peptides were not isolated when [2-3H]8-N3-Ado was incubated with apo AdoHcy hydrolase and irradiated. The two photolabeled peptides are assumed to be parts of the adenine-binding domain for substrates. They are both within well conserved regions of AdoHcy hydrolases. The peptide Val175-Lys186 is located very close to Cys195 and Glu197. Ser198, both of which were indicated to be located in the active site of the enzyme by chemical modification and limited proteolysis methods. The peptide Val319-Arg327 is adjacent to Leu330, which is proposed by a computer graphics model to interact with the C-6-NH2 group of Ado.