We recently showed HLA-B35-restricted CTL activity for 10 HIV-1 epitopes in PBL from two HIV-1-infected individuals. In the present study, we established CTL clones specific for nine of these HIV-1 epitopes to confirm these HLA-B35-restricted epitopes. The specific CTL clones effectively killed the HLA-B*3501-positive target cells infected with the HIV-1 vaccinia recombinant virus. These results confirmed that nine HIV-1 CTL epitopes are presented by HLA-B*3501 molecules. The CTL activity specific for four Pol and two Nef epitopes was induced in the peptide-stimulated PBL from three or more of seven HIV-1-infected individuals, indicating that these six are common epitopes. Eight were considered strong epitopes because the specific CTL activity was detected in the cultured PBL that was once stimulated with peptides. Thus, the present study excluded the possibility that the disability of the presentation of HIV-1 epitopes by HLA-B35 molecules is associated with the accelerated progression of AIDS in HLA-B35-positive individuals. Analysis of mutated epitopes found in an HIV-1 type B strain using the CTL clones revealed that most mutated epitopes partially or markedly affect the recognition of CTL clones. Of 19 mutations that affected recognition of the CTL clones, 7 reduced peptide-HLA-B*3501 binding, while 12 affected TCR recognition. These results indicate that natural mutations of HLA-B35-restricted HIV-1 CTL epitopes affect the recognition of CTL by mechanisms that reduce both peptide binding and TCR recognition.