Human immunodeficiency virus type 1 (HIV-1) Gag-specific cytotoxic T lymphocyte (CTL) responses were studied in seven seropositive long-term asymptomatic individuals (CDC A1) with stable CD4 counts for more than 8 years. Using a set of partially overlapping peptides covering the whole Gag, five 15-20-mer peptides were found to contain CTL epitopes. Further characterization of these epitopes revealed a new HLA-A25-restricted CTL epitope in p24, p24(203-212) ETINEEAAEW. This region of Gag is highly conserved in clades B and D of HIV-1. Naturally occurring amino acid sequences, containing p24(203)D (consensus HIV-1 clades A, C, F, G and H) or p24(204)I (HIV-2ROD) were not recognized by CTL recognizing the index peptide. No virus variants with mutations in this sequence were found in peripheral blood mononuclear cells from the HIV-1-infected individual concerned during the 8 year observation period, indicating that the virus had not escaped from the observed CTL response.