In human immunodeficiency virus (HIV) infection, sequence variations within defined cytotoxic T lymphocyte (CTL) epitopes may lead to escape from CTL recognition. In a previous report, we have shown that the variable central region of HIV Nef protein (amino acids 73-144) that contains potential CTL epitopes, can escape the CTL response. We suggested that this non recognition occurs through a variety of mechanisms. In particular, we provided evidence that HIV Nef sequences recovered from HLA-A11-expressing individuals have alterations in the major anchor residues essential for binding of the two Nef epitopes (amino acids 73-82 and 84-92) to the HLA-A11 molecule. Here, we investigate in more detail whether variations in autologous Nef sequences affect HLA binding, leading to CTL escape. Potential epitopes were sought by testing Nef peptides containing the HLA-A11-specific motif or related motifs. We confirmed that only the two previously described epitopes identified in cytolysis tests have optimal reactivity with the HLA-A11 molecule. We then sequenced several viral variants from donors that do not express the HLA-A11 molecule and compared the variability of these epitopes with those obtained from HLA-A11-expressing individuals. One substitution (Leu85) found in the sequences isolated from both populations increase the reactivity of the HLA-A11-restricted epitope 84-92, and might explain the difference in immunogenicity observed between the two HLA-A11-restricted epitopes from HLA-A11+ individuals. In addition, selective variations were only detected in virus isolated from HLA-A11-expressing individuals. Furthermore, examination of the association of variant peptides with the HLA-A11 molecule demonstrated that a single substitution within the minimal epitope could not always completely abrogate HLA binding, suggesting that multiple alterations within a particular epitope may accumulate during disease progression, allowing the virus to escape CTL recognition.