The HIV-1 regulatory proteins Tat and Rev and the accessory proteins Vpr, Vpu, and Vif are essential for viral replication, and their cytoplasmic production suggests that they should be processed for recognition by cytotoxic T lymphocytes. However, only limited data is available evaluating to which extent these proteins are targeted in natural infection and optimal cytotoxic T lymphocyte (CTL) epitopes within these proteins have not been defined. In this study, CTL responses against HIV-1 Tat, Rev, Vpr, Vpu, and Vif were analyzed in 70 HIV-1 infected individuals and 10 HIV-1 negative controls using overlapping peptides spanning the entire proteins. Peptide-specific interferon-gamma (IFN-gamma) production was measured by Elispot assay and flow-based intracellular cytokine quantification. HLA class I restriction and cytotoxic activity were confirmed after isolation of peptide-specific CD8+ T-cell lines. All regulatory and accessory proteins served as targets for HIV-1- specific CTL and multiple CTL epitopes were identified in functionally important regions of these proteins. In certain individuals HIV-1-specific CD8+ T-cell responses to these accessory and regulatory proteins contributed up to a third to the magnitude of the total HIV-1-specific CTL response. These data indicate that despite the small size of these proteins regulatory and accessory proteins are targeted by CTL in natural HIV-1 infection, and contribute importantly to the total HIV-1-specific CD8+ T-cell responses. These findings are relevant for the evaluation of the specificity and breadth of immune responses during acute and chronic#10; infection, and will be useful for the design and testing of candidate human immunodeficiency virus (HIV) vaccines.