Recent single-molecule Förster resonance energy transfer studies of DNA polymerase I have led to the proposal of a postinsertion fidelity-checking site. This site is hypothesized to ensure proper base pairing of the newly inserted nucleotide. To help test this hypothesis, we have used energy decomposition, electrostatic free energy response, and noncovalent interaction analysis analyses to identify residues involved in this putative checking site. We have used structures of DNA polymerase I from two different organisms, the Klenow fragment from Escherichia coli and the Bacillus fragment from Bacillus stearothermophilus. Our results point to several residues that show altered interactions for three mispairs compared to the correctly paired DNA dimer. Furthermore, many of these residues are conserved among A family polymerases. The identified residues provide potential targets for mutagenesis studies for investigation of the fidelity-checking site hypothesis.