We used SIV infection of macaques as an animal model to assess DNA immunization as a strategy for protecting against HIV infection in humans. We compared the efficacy of DNA immunization alone and in combination with subunit protein boosts. The DNA vaccine consisted of all structural and regulatory genes of SIVmne cloned into mammalian expression vectors. Eight cynomolgus macaques were immunized twice, at weeks 0 and 8, with plasmid DNA and four control animals received vector DNA alone. Four primed macaques also received DNA boosters at weeks 16 and 36, while the second group of four were boosted with recombinant Env plus recombinant Gag-Pol particles in oil emulsion adjuvant. Two controls received vector-only DNA and two received adjuvant alone. As expected, humoral immune responses were stronger in the macaques that received subunit boosts, but responses were sustained in both groups, as measured by ELISA antibody titers to SIV Env. We measured neutralizing antibody titers, cytotoxic T lymphocyte responses, and T cell proliferative responses in all vaccinees over the course of immunization. All animals were challenged at week 38 with SIVmne by the intrarectal route. Post-challenge virus load was measured in all animals and there were major differences between the groups in the challenge outcome. Surprisingly, sustained low virus loads were observed only in the DNA group (2/4 macaques), suggesting that 4 immunizations with DNA alone elicited more effective immune responses than 2 DNA primes combined with 2 protein boosts. This study represents the first case of DNA immunization eliciting protective immunity against a pathogenic SIV challenge. Multigenic DNA vaccines such as these, bearing all structural and regulatory genes, show significant promise and may be a safe alternative to live-attenuated vaccines.