The substrate deoxynucleoside triphosphate (dNTP) binding site of Moloney murine leukemia virus (M-MuLV) reverse transcriptase was labeled with pyridoxal 5'-phosphate (PLP), a substrate binding site-directed reagent for DNA polymerases (Modak, M. J. (1976) Biochemistry 15, 3620-3626). Treatment of M-MuLV reverse transcriptase with PLP results in the loss of RNA-dependent DNA polymerase activity, but has no effect on ribonuclease H activity. Neither template-primer nor substrate dNTP alone shows any protective effect from PLP-mediated inactivation. However, the presence of both template-primer and complementary substrate dNTP significantly protects M-MuLV reverse transcriptase from PLP inhibition. Using tritiated sodium borohydride to label the pyridoxylated enzyme, approximately 4 mol of PLP were incorporated per mol of enzyme. In the presence of template-primer and the complementary dNTP, however, only 2 mol of PLP were incorporated. Comparative tryptic peptide mapping of enzyme, modified in the presence and absence of substrates by PLP reaction on C-18 reverse phase columns, indicated the protection of two peptides from pyridoxylation in the presence of substrate triphosphate. These two peptides were further purified and characterized by amino acid analyses and sequencing and were found to span residues 103 to 110 and 412 to 425 in the primary amino acid sequence of M-MuLV reverse transcriptase. Furthermore, Lys-103 of peptide I and Lys-421 of peptide II were found to be the targets of pyridoxylation, indicating that these 2 lysine residues are involved in substrate dNTP binding in M-MuLV reverse transcriptase.