Polymerases in general share only a few regions of amino acid similarity. One of the most conserved regions, called motif A, has the sequence DXXSLYPSII or a similar sequence in many eukaryotic and viral DNA polymerases and in bacteriophage T4 DNA polymerase. We designed genetic techniques to isolate mutant T4 DNA polymerases with amino acid substitutions in this highly conserved motif. The mutant DNA polymerases differed from wild type T4 DNA polymerase in several ways. For one mutant DNA polymerase, the pyrophosphate analog, phosphonoacetic acid, was a potent inhibitor of DNA replication, and this mutant DNA polymerase replicated DNA with reduced fidelity. Another mutant DNA polymerase replicated DNA with increased accuracy, but this mutant DNA polymerase was less processive in primer extension reactions, and DNA replication required high concentrations of deoxynucleoside triphosphates. We provide evidence that indicates that all of these changes to DNA polymerase function are due to differences in how the mutant DNA polymerases partition between states active for DNA replication or exonucleolytic proofreading. These studies also provide further support for the hypothesis that the accuracy of DNA replication observed for DNA polymerases and 3'-->5' exonuclease activities (Muzyczka, N., Poland, R. L., and Bessman, M. J. (1972) J. Biol. Chem. 247, 7116-7122).