Sixty percent of rhesus macaques infected with virulence attenuated virus SHIV 89.6 are protected from subsequent intravaginal challenge with pathogenic SIVmac239 [Abel K, Compton L, Rourke T, Montefiori D, Lu D, Rothaeusler K, et al. Simian-human immunodeficiency virus SHIV89.6-induced protection against intravaginal challenge with pathogenic SIVmac239 is independent of the route of immunization and is associated with a combination of cytotoxic T-lymphocyte and alpha interferon responses. J Virol 2003;77(5):3099-118; Miller CJ, McChesney MB, Lu X, Dailey PJ, Chutkowski C, Lu D, et al. Rhesus macaques previously infected with simian/human immunodeficiency virus (HIV) are protected from vaginal challenge with pathogenic SIVmac239. J Virol 1997;71(3):1911-21]. Previously, we have shown that inoculation with a proviral plasmid encoding SHIV 89.6 (pMA SHIV-89.6) results in systemic infection that is delayed compared to SHIV 89.6 virus inoculation [Busch M, Lu D, Fritts L, Lifson JD, Miller CJ. Comparison of virology and immunology in SHIV 89.6 proviral DNA and virus-inoculated rhesus macaques. J Med Primatol 2003;32(4-5):240-6]. We now report that, although monkeys inoculated with pMA SHIV-89.6 or SHIV 89.6 virus had similar plasma anti-SIV binding antibody titers and number of anti-SIV IFN-gamma secreting cells on the day of mucosal SIVmac239 challenge, a smaller proportion of monkeys immunized with pMA SHIV-89.6 were protected from vaginal SIVmac239 challenge compared to monkeys immunized using SHIV 89.6 virus. Protected DNA immunized monkeys had stronger anti-SIV IFN-gamma ELISPOT responses in the acute stage post-challenge than unprotected monkeys. Plasma anti-SIV binding antibody titers and PBMC cytokine responses in the acute stages post-challenge were similar in DNA vaccinated-protected and DNA vaccinated-unprotected monkeys. These results suggest that the delay in systemic infection resulting from delivery of SHIV 89.6 as a plasmid decreased the effectiveness of this live attenuated vaccine.