In this investigation we have examined the effect of human interferon (IFN) type alpha on the ability of vaccinia virus to recombine within infected African green monkey kidney cells (BSC-40). We measured by marker rescue, the extent of insertion of cloned 5-kb Hind III-J restriction fragment of wild-type vaccinia DNA into the genome of temperature-sensitive mutants. We showed that IFN at doses of 100-1000 U/ml inhibited the replication of vesicular stomatitis virus (VSV) and polio viruses but not of vaccinia virus. Vaccinia virus adsorption, penetration, uncoating, protein synthesis, and yields were not inhibited. However, marker rescue of vaccinia virus was inhibited by IFN. This inhibition was not related to IFN-mediated changes in uptake of exogenous DNA or enhanced degradation of the transfected DNA. These results suggest that IFN affects homologous vaccinia DNA recombination.