DNA polymerase exonucleolytic proofreading is important in attaining high fidelity DNA replication. One of the most well characterized proofreading activities is the 3'-->5'-exonuclease activity of bacteriophage T4 DNA polymerase. We have used genetic analyses and protein sequence comparisons to Escherichia coli DNA polymerase I to identify amino acids in the N-terminal region of T4 DNA polymerase that are required for exonucleolytic proofreading. Mutant DNA polymerases with amino acid substitutions D112A/E114A, D219A, or D324A reduced 3'-->5'-exonuclease activity 10(2)-10(4)-fold in various in vitro assays and decreased DNA replication fidelity in vivo. DNA replication activity was also reduced for the exonuclease-deficient DNA polymerases in vitro and in vivo. Reduction in DNA replication appeared to be due primarily to the interdependence of T4 DNA polymerase replication and proofreading activities; T4 DNA polymerase requires 3'-->5'-exonuclease activity to repair primer termini that are not suitable substrates for extension. Observations reported here provide further evidence in support of the proposal that DNA polymerases have distinct 3'-->5'-exonuclease and polymerase active sites.