Generation of cytotoxic-T-lymphocyte (CTL) responses against mutated ras peptides from peripheral-blood mononuclear cells (PBMC) was attempted in a group of HIA-A2.I+ healthy donors. Bulk PBMC cultures were stimulated in vitro with a mixture of peptides encompassing I 2 Gly + Val, 6 I Gln + Lys or 6 I Gln + Leu ras mutations and displaying HIA-A2. I binding motifs, selected by a computer program. A promiscuous tetanus toxoid peptide was also added. Weekly thereafter, PBMC were re-stimulated with peptide pulsed autologous Epstein-Barrvirus (EBV)-transformed B cells. After 8 rounds of re-stimulation, reproducible cytotoxic activity against peptide-pulsed target cells was detectable in one donor. The CTL line recognized 2 nonamers encompassing ras 61 Gln + Leu mutation. Killing was mediated by CD8+ T cells displaying 4 TCR and was inhibited by anti-HIA-N. I monoclonal antibodies. N o killing of tumor cells expressing the specific mutation could be observed. More than 60 CTL clones were generated. Fine specificity studies revealed effective, though differing cytotoxic activity against both 53-LDILDTAGL-6 I and 55-ILDTAGLEE-63, but not against 54-DILDTAGLE-62 mutated peptides, in all but one of the clones. None was able to exert effective cytotoxic activity against tumor cells expressing the specific mutation. T-cellreceptor (TCR) usage was then analyzed phenotypically, by reverse-transcription-polymerase-chain-reaction (RT-PCR) and by sequence analysis. This study revealed the monoclonal nature of the CTL response against mutated nonamers, with TCR expressing Vp14 gene product in combination with, J82.7 and C82.