Summary Nineteen recombinants between vaccinia virus (VV) DNA- temperature-sensitive mutants and ectromelia virus (EMV) were characterized with respect to their biological properties and genome structure. Four of these recombinants acquired the pathogenicity for mice characteristic of EMV, while the pathogenicity for rabbits characteristic of VV was not only preserved, but even enhanced. Most unexpectedly, these recombinants with ‘double pathogenicity’, as well as four other recombinants, acquired a stable genetic trait which was not typical of the parental viruses, i.e. the ability to form haemorrhagic lesions on the chorioallantoic membrane of chick embryos. Approximate mapping of the genomes of these recombinants with restriction endonucleases showed that their DNA contained mostly VV sequences with a single detected insert of EMV DNA. In the ‘double pathogenicity’ recombinants, this insert was located in the central part of the genome, and its minimal size was about 16 Mdal.