Using the technique of UV-mediated cross-linking of nucleotides to their acceptor sites (Modak, M. J., and Gillerman-Cox, E. (1982) J. Biol. Chem. 257, 15105-15109), we have labeled calf terminal deoxynucleotidyltransferase (TdT) with [32P]dTTP. The specificity of dTTP cross-linking at the substrate binding site in TdT is demonstrated by the competitive inhibition of the cross-linking reaction by other deoxynucleoside triphosphates, and ATP and its analogues, requiring concentrations consistent with their kinetic constants. Tryptic peptide mapping of the [32P]dTTP-labeled enzyme showed the presence of a single radioactive peptide fraction that contained the site of dTTP cross-linking. The amino acid composition and sequence analysis of the radioactive peptide fraction revealed it to contain two tryptic peptides, spanning residues 221-231 and 234-249. Since these two peptides were covalently linked to dTTP, the region encompassed by them constitutes a substrate binding domain in TdT. Further proteolytic digestion of the tryptic peptide-dTTP complex, using V8 protease, yielded a smaller peptide, and its analysis narrowed the substrate binding domain to 14 amino acids corresponding to residues 224-237 in the primary amino acid sequence of TdT. Furthermore, 2 cysteine residues, Cys-227 and Cys-234, within this domain were found to be involved in the cross-linking of dTTP, suggesting their participation in the process of substrate binding in TdT.