Post-vaccinial encephalitis and disseminated vaccinia are major concerns with the use of vaccinia virus recombinants as immunization vectors in man. To identify and characterize possible attenuated poxvirus vectors, rabbitpox virus (RPV) (closely related to vaccinia) and four deletion mutants of RPV were studied for organ tropism, neurovirulence, and protection from wild-type challenge in BALB/c mice. Intraperitoneal (IP) inoculation with 10(7) PFU wild-type (wt) RPV or with two mutants 8 sm and 28 (containing approximately 12 kilobase deletions) showed titers of greater than 10(3) PFU/g tissue in multiple organs. In contrast, IP inoculation of 10(7) PFU of mutants 31 or 23 (containing approximately 30 kilobase deletions) showed markedly reduced growth in all organs. Neurovirulence of wt and mutant RPV was determined by intracerebral (IC) inoculation of mice. Wt and mutants 8 sm, and 28 RPV had LD50 less than 10(2) PFU; in contrast, 31 and 23 had LD50 greater than 10(5) PFU. Finally, 10(6) PFU of mutants 31 or 23, were administered to mice by scarification, the normal route of vaccinia immunization. Both 31 and 23 grew locally in the skin and protected mice challenged IC at 21 days with 100 LD50 of wt RPV, while all unimmunized controls died. We conclude that deletion mutants 31 and 23 demonstrate markedly reduced invasiveness and neurovirulence while retaining immunogenicity. Similar deletion mutations in vaccinia may create avirulent, but effective vaccine vectors for man.