Pyridoxal 5'-phosphate (PLP) is an inhibitor of DNA polymerase activity of Escherichia coli DNA polymerase I large fragment. Kinetic studies indicated that overall PLP inhibition was noncompetitive with respect to dNTP, and Hill plot analysis revealed that two molecules of PLP were involved in the inhibition. Reduction of the PLP-treated enzyme with sodium [3H]borohydride resulted in covalent incorporation of 3 mol of PLP/mol of enzyme. This incorporation was at lysine residues exclusively, and the PLP-modified enzyme was not capable of DNA polymerase activity. The presence of dNTP during the modification reaction blocked the incorporation of 1 mol of PLP/mol of enzyme. Similar results were obtained in the presence or absence of template-primer. These data indicate that a PLP target lysine is in or around a dNTP binding site that is essential for polymerase activity and that this binding site is functional in the absence of template-primer. The enzyme modified in the presence of dNTP, containing 2 mol of PLP/mol of enzyme, was capable of DNA polymerase activity but was unable to conduct elongation of product molecules beyond a short oligonucleotide length.