Transferred nuclear Overhauser effects (NOEs) and selective T1 measurements were used to determine interproton distances in the substrates Mg2+dATP and Mg2+TTP bound to the large fragment of DNA polymerase I (Pol I). The distances are consistent with high anti, O1' endo conformations for the enzyme-bound substrates, similar to nucleotides of B-DNA. These substrate conformations show little or no change when the complementary RNA templates (rU)57 or (rA)50 are bound. In contrast, multiple conformations, including syn and anti species, are required to fit the interproton distances measured on the enzyme-bound guanine nucleotide substrates Mg2+dGTP and Mg2+ddGTP. These multiple substrate conformations simplify to a single high anti, O1' endo conformation when the complementary template (rC)37 is bound, possibly due to base-pairing with the template, as in the active complex. In the presence of both template and primer, enzyme-bound Mg2+ddGTP reverts to multiple conformations. This ability of Pol I to decrease the fraction of bound substrate which is appropriate for primer elongation may be an error-preventing mechanism. In all cases, the conformations of the average nucleotide of the enzyme-bound RNA templates are also B-like. Transferred NOEs from protons of the enzyme to those of bound dNTP substrates suggest hydrophobic (Ile, Leu) and an aromatic amino acid (Tyr) at the substrate binding site. Peptide I, a synthetic 50-residue peptide based on residues 728 to 777 of the Pol I sequence, containing the conserved sequence L-I-Y-G, retains significant secondary and tertiary structure in solution as found by circular dichroism (CD) and 2D NMR. While the X-ray structure shows 48% helix in this region, the sequence specific NOESY analysis suggests 18% helix, and the preservation of two of the three beta turns. Peptide I shows tight binding of dNTP substrates, the substrate analog 2',3'-trinitrophenyl-ATP, and duplex DNA, providing direct evidence that the active site for polymerization lies in this region of the enzyme, with the substrate binding along the O-helix near Leu-764, Ile-765, and Tyr-766. Another synthetic peptide, peptide II, based on residues 840 to 888 of the Pol I sequence also retains much secondary structure as detected by CD but does not bind the substrate analog TNP-ATP.