The fidelity of homologous recombination in vaccinia virus (VV) DNA was examined by constructing a viral recombinant whose genome contained a copy of the Escherichia coli lac z gene in which the central third of the gene was repeated on either side of the VV thymidine kinase (tk) gene. In this virus, homologous DNA recombination and consequent excision of the tk gene were necessary to restore the open reading frame for beta-galactosidase and thereby to confer a lac z+ phenotype. Imprecise recombination was predicted to increase the frequency of lac z- virus. However, after several passages during which almost every viral genome underwent homologous recombination, the frequency of lac z- plaques was indistinguishable from that of a control virus that could express beta-galactosidase without prior recombination. We conclude that homologous recombination in VV DNA occurs with perfect fidelity at least 99% of the time.