The base substitution fidelity of DNA polymerase-alpha, -beta, and -gamma (pol-alpha, -beta, and -gamma, respectively) has been determined in vitro, for all 12 possible mispairs at 96 sites in a forward mutational target. Averaging all errors over all known detectable sites, pol-gamma is the most accurate enzyme, producing one error for every 10,000 bases polymerized. Pol-beta is much less accurate, with an error rate of 1/1,500, while pol-alpha has an intermediate accuracy of 1/4,000. The relative differences in fidelity between the DNA polymerases are strongly influenced by the nature of the mispair. For example, G(template):dATP mispairs and G:dGTP mispairs are formed with about equal frequency by all three classes of DNA polymerases, yet pol-gamma produces T:dGTP mispairs at a 100-fold lower frequency than does pol-beta. The DNA polymerases exhibit distinct differences in template site preferences as well as substrate insertion preferences. The increase in accuracy apparent in proceeding from the least selective to the most accurate enzyme results primarily from a decrease in mispair formations at template A and T residues and a decrease in misinsertion of pyrimidine deoxynucleotides. These data clearly demonstrate a major role for eucaryotic DNA polymerases in modulating base mispair frequencies at the level of insertion. In addition to direct mispair formation due to an incorrect incorporation event, an examination of the errors produced by each of the three classes of DNA polymerases at two particular sites in the target sequence suggests that some base substitution errors result from transient misalignment of the primer-template. A model is presented to explain this phenomenon, termed "Dislocation Mutagenesis." The data are discussed in relation to the extensive literature on base substitution errors and to the origin of spontaneous base substitutions in animal cells.