The phi X174 fidelity system provides a biological assay for quantitating the accuracy of DNA polymerases. Expansion of this system to cell extracts and DNA replication complexes from eucaryotes has been limited by the presence of nucleases in these preparations. We have overcome these limitations by priming the phi X template with a synthetic oligodeoxynucleotide, with its free 3'-hydroxyl terminus only a short distance from the amber locus that is the site for determining the frequency of misincorporation. In this paper, this modified phi X system is characterized and compared to that using defined natural DNA restriction fragments as primers. The modified system has been applied to studies on the fidelity of DNA synthesis using different forms of purified DNA polymerase-alpha from calf thymus, as well as crude extracts from human lymphocytes.