The impact of HIV-1 Nef-mediated HLA-I down-regulation on CD8(+) cytotoxic T lymphocytes (CTLs) varies by epitope, but the determining factors have not been elucidated. In the present study, we investigated the impact of Nef on the antiviral efficiency of HIV-1-specific CTLs targeting 17 different epitopes to define properties that determine susceptibility to Nef. The impact of Nef was not correlated with the presenting HLA-I type or functional avidity of CTLs, but instead was related directly to the kinetics of infected cell clearance. Whereas Gag-specific CTLs generally were less susceptible to Nef than those targeting other proteins, this was determined by the ability to eliminate infected cells before de novo synthesis of viral proteins, which was also observed for CTLs targeting a Nef epitope. This very early clearance of infected cells depended on virus inoculum, and the required inoculum varied by epitope. These results suggest that whereas Gag-specific CTLs are more likely to recognize infected cells before Nef-mediated HLA-I down-regulation, this varies depending on the specific epitope and virus inoculum. Reduced susceptibility to Nef therefore may contribute to the overall association of Gag-specific CTL responses to better immune control if a sufficient multiplicity of infection is attained in vivo, but this property is not unique to Gag.