In an attempt to understand the molecular mechanisms of spontaneous and induced mutagenesis in higher organisms, the mutational specificities of highly purified DNA polymerases alpha, beta and gamma have been assessed for a single round on in vitro DNA synthesis with undamaged, biologically active DNA. A forward mutational assay in a nonessential gene has been used, which is capable of detecting frameshift and deletion errors in addition to all twelve possible base substitution errors at 96 different positions in a 250 base target sequence (the lacZ alpha gene in M13mp2 DNA). DNA sequence analyses of over 1000 mutants generated by these enzymes demonstrate the production in vitro of all three classes of errors. The frequencies and specificities of these mutations are highly distinctive for each class of DNA polymerase. These data point to properties of both the DNA and the enzymes themselves which are important in determining the frequency and specificity of spontaneous and perhaps induced mutagenesis.