The fidelity of DNA synthesis as determined by the misincorporation of the base analogue 2-aminopurine in competition with adenine has been measured as a function of deoxynucleoside triphosphate substrate concentrations using purified mutator (L56), antimutator (L141), and wild type (T4D) T4 DNA polymerases. Although the rates of both incorporation and turnover of aminopurine and adenine decrease as substrate concentrations are decreased, the ratio of turnover/polymerase activity is increased. Thus, the nuclease/polymerase ratio of each of these three DNA polymerases can be controlled. The misincorporation of aminopurine decreases with decreasing substrate concentrations such that all three enzymes approach nearly identical misincorporation frequencies at the lowest substrate concentration. The increased accuracy of DNA synthesis corresponds to conditions producing a high nuclease/polymerase ratio. The misinsertion frequency for aminopurine is independent of substrate concentrations and enzyme phenotype; therefore, the increased accuracy of DNA synthesis with decreasing substrate concentrations is shown to be a result of increased nuclease activity and not increased polymerase or nuclease specificity. The data are analyzed in terms of a kinetic model of DNA polymerase accuracy which proposes that discrimination in nucleotide insertion and removal is based on the free energy difference between matched and mismatched base pairs. A value of 1.1 kcal/mol free energy difference, delta G, between adenine: thymine and aminopurine:thymine base pairs is predicted by model analysis of the cocentration dependence of aminopurine misincorporation and removal frequencies. An independent estimate of this free energy difference based on the 6-fold higher apparent Km of T4 DNA polymerase for aminopurine compared to adenine also gives a value of 1.1 kcal/mol. It is shown that the aminopurine misinsertion frequency for an enzyme having either extremely low 3'-exonuclease activity, Escherichia coli DNA polymerase I, or no measurable exonuclease activity, calf thymus DNA polymerase alpha, is 12 to 15%, which is similar to that for the T4 polymerases and consistent with delta G approximately 1.1 kcal/mol.