Given the current difficulties generating vaccine-induced neutralizing antibodies to human immunodeficiency virus (HIV), the focus of the vaccine community has shifted toward creating cytotoxic-T-lymphocyte (CTL)-based vaccines. Recent reports of CTL-based vaccine trials in macaques challenged with simian/human immunodeficiency virus SHIV-89.6P have supported the notion that such vaccines can ameliorate the course of disease. However, almost all of these studies included Env as an immunogen and since SHIV-89.6P is sensitive to neutralizing antibodies it is difficult to determine the mechanism(s) of protection. Consequently, SHIV-89.6P challenge of macaques may be a poor model for determining vaccine efficacy in humans. To ascertain the effect of vaccine-induced multispecific mucosal CTL, in the absence of Env-specific antibody, on the control of an immunodeficiency virus challenge, we vaccinated Mamu-A*01(+) macaques with constructs encoding a combination of CTL epitopes and full-length proteins (Tat, Rev, and Nef) by using a DNA prime/recombinant modified vaccinia virus Ankara (rMVA) boost regimen. The vaccination induced virus-specific CTL and CD4(+) helper T lymphocytes with CTL frequencies as high as 20,000/million peripheral blood mononuclear cells. The final rMVA vaccination, delivered intravenously, engendered long-lived mucosal CTL. At 16 weeks after the final rMVA vaccination, the vaccinees and naive, Mamu-A*01(+) controls were challenged intrarectally with SIVmac239. Massive early anamnestic cellular immune responses controlled acute-phase viral replication; however, the three vaccinees were unable to control virus replication in the chronic phase. The present study suggests that multispecific mucosal CTL, in the absence of neutralizing antibodies, can achieve a modicum of control over early viral replication but are unable to control chronic-phase viral replication after a high-dose mucosal challenge with a pathogenic simian immunodeficiency virus.