DNA vaccination of rabbit skin with the L1 gene of cottontail rabbit papillomavirus (CRPV) has previously been shown to induce prophylactic immunity against CRPV. We now describe the effects of vaccination with the CRPV E6 gene, using the same approach. The experimental vaccine pdCMV-E6 encoded both the truncated and full length forms of CRPV E6 protein. The control vaccine pCMV-beta encoded beta galactosidase. Rabbits were vaccinated with DNA-coated gold particles, using a gene gun. Each rabbit received an initial vaccination with 30 micrograms DNA and 3 weeks later a booster vaccination, also with 30 micrograms DNA. pdCMV-E6-vaccinated rabbits developed E6-specific cellular immunity as determined by proliferation assays using peripheral blood mononuclear cells from animals prior to challenge, but did not develop detectable humoral immunity to E6 proteins, as evaluated by ELISA using two different E6 antigen preparations. Control rabbits developed humoral immunity to beta galactosidase. All rabbits were challenged by infection of nine skin sites with live CRPV virus and monitored for papilloma formation. None of four control rabbits was protected at any of the challenge sites. Of six rabbits vaccinated with pdCMV-E6, two were completely protected and one was virtually completely protected (tiny papillomas at just two of nine challenge sites). These three rabbits also exhibited significant E6-specific in vitro proliferative responses. The four E6 DNA-vaccinated rabbits that were not completely protected exhibited evidence of partial protection: some challenge sites did not form papillomas; papilloma onset was delayed; papilloma burden was less. These results demonstrate that partial prophylaxis against papillomavirus-induced disease can be achieved by intracutaneous vaccination with a recombinant plasmid encoding the papillomavirus.