The fidelity with which wild type T4 DNA polymerase copies phi X174 amber 3 plus strand DNA at position 587 in vitro has been measured. Synthesis is initiated by hybridizing to the template a HaeIII restriction fragment whose 3'-OH terminus is 83 nucleotides from the amber 3 site. Based on gel electrophoresis of product DNA molecules and genetic marker rescue data, T4 DNA polymerase copies significantly beyond the mutant site. Transfection analysis shows that the A X T leads to G X C mutation at position 587 occurs 10- to 100-fold less frequently with T4 DNA polymerase than with E. coli DNA polymerase I. The aberrant incorporation of cytosine opposite adenine at position 587 by the T4 polymerase alone is occurring at a frequency not greater than about 10(-7) which, for this particular locus, may be similar to the fidelity exhibited by the T4 accessory proteins plus the polymerase comprising the replication complex. A comparison of the accuracy of mutator L56 and antimutator L141 T4 DNA polymerases relative to wild type shows at most a 2- to 4-fold decrease and increase, respectively, in fidelity. When compared to 10- to 1000-fold effects on mutation frequencies that these same mutant alleles have in vivo, these results suggest that the wide range in expression of mutator and antimutator phenotypes in vivo may be dependent on an abnormal interaction of the aberrant DNA polymerases with other protein components of the replication complex.