The wild-type (WT) vaccinia (WR strain) virus is highly virulent to mice by intranasal inoculation, yet death can be prevented by cidofovir treatment. A cidofovir-resistant (CDV-R) mutant of the virus was developed by 15 Vero cell culture passages in order to determine cross-resistance to other inhibitors, growth characteristics, virulence in infected mice, and suitability of the animal model for studying antiviral therapies. Comparisons were made to the original WT virus and to a WT virus passaged 15 times in culture (WTp15 virus). Cidofovir inhibited WT, WTp15, and CDV-R viruses by 50% at 61, 56 and 790 μM, respectively, in plaque reduction assays, with similar inhibition seen in virus yield studies. Cross-resistance occurred with compounds related to cidofovir, but not with unrelated nucleosides. The resistant virus produced 300-fold fewer infectious particles (PFU) than WT and WTp15 viruses in mouse C127I cells, yet replicated similarly in Vero (monkey) cells. The CDV-R virus was completely attenuated for virulence at 107 PFU per mouse in normal BALB/c mice and in severe combined immunodeficient (SCID) mice. The WTp15 virus was 100-fold less virulent than WT virus in BALB/c mice. Thus, the lack of virulence of the resistant virus in the animal model is explained partly by its reduced ability to replicate in mouse cells and by attenuation occurring as a result of extensive cell culturing (inferred from what occurred with the WTp15 virus). Lung and snout virus titre reduction parameters were used to assess antiviral activity of compounds in BALB/c mice infected intranasally with the CDV-R virus. Cidofovir, HDP-cidofovir and arabinofuranosyladenine treatments reduced lung virus titres.