The high fidelity of the DNA polymerization process is critically important for the stability of the cellular genome. The role of template and incoming nucleotide base pairing in polymerase fidelity has recently been explored by the use of nucleotide isosteres, which preserve the steric but not the electronic properties of the corresponding bases. The DNA repair enzyme, DNA polymerase beta (Pol beta), is among the most discriminating, being inactive when the thymine isostere difluorotoluene (DFT) is present in the templating base position. To explore the physical basis for this inactivity, we have performed NMR studies on [methyl-13C]methionine-labeled Pol beta complexed with double-hairpin DNA, used to model the gapped nucleotide substrate, and having either a thymine or a DFT isostere at the templating base position. The six methionine residues distributed throughout the enzyme provide useful conformational probes of the lyase and polymerase domains and subdomains. Analysis of the proton shift of Met282 that results from formation of an abortive Pol beta-gapped DNA-dATP complex is consistent with an open to closed conformational change of the enzyme predicted from crystal structures. In contrast, the same resonance is nearly unshifted when a ternary complex is formed from dATP and gapped DNA in which a DFT isostere replaces thymine at the templating base position. Alternatively, the resonances of Met191 and Met155, located in the catalytic subdomain, show perturbations upon formation of the abortive ternary complex, which are qualitatively similar, but significantly weaker, than the changes observed when thymine is present at the templating base position. The changes in the Met155 and Met191 methyl resonances are in fact more similar to those observed in the binary Pol beta-dATP complex. These studies demonstrate that the block in catalysis is directly related to the absence of the set of conformational transitions that include the "open" to "closed" transition monitored by Met282.