To further our understanding of the structure and function of the vaccinia virus DNA polymerase, we have performed fine genetic analysis of three mutants with lesions in the polymerase gene. By performing marker rescue analysis with DNA fragments of decreasing size, each lesion was localized to within 500 base pairs of DNA. The relevant regions of the mutant alleles were then cloned and subjected to DNA sequence analysis, which allowed the assignment of a single nucleotide and amino acid change to each mutant. As well as providing structure-function correlations germane to an understanding of polymerase activity, these data have provided insights into the frequency and possible mechanisms of viral homologous recombination.