Abstract In this study, we examined the role of simian immunodeficiency virus (SIV)-specific cytotoxic T lymphocytes (CTLs) in macaques immunized with an attenuated strain of simian immunodeficiency virus (SIVmac239Δnef) in protection against pathogenic challenge with SIVmac251. Our results indicate that attenuated SIVmac239Δnef can elicit specific CTL precursor cells (CTLp), but no correlation was observed between breadth or strength of CTLp response to structural proteins SIV-Env, -Gamg or -Pol (as measured by limiting dilution assay) and protection against infection. In one animal, we longitudinally followed the SIV-Gag-specific response to an MHC class I Mamu-A*01-restricted epitope p11C, C-M using a tetrameric MHC/peptide complex reagent. A low frequency of SIV p11C, C-M peptide-specific tetramer-reactive cells was present at the time of challenge but could be expanded in vitro. Surprisingly, the low level of Mamu-A*01/p11C, C-M-specific CTLs induced through attenuated SIVmac239Δnef vaccination increased in the absence of detectable SIVmac251 or SIVmac239Δnef proviral DNA. Overall, our results suggest that protection against infection in this model can be achieved through more than one mechanism, with SIV-specific CTLs being important in controlling SIVmac239Δnef viral replication postchallenge.