Activated ras carry a point mutation either in codon 12, 13 or 61 which is tumor specific. Peptides derived from this oncoprotein are therefore potential tumor antigens. Essential for the feasibility of using ras-derived peptides in therapy of cancer is whether p21 ras-derived peptides can be processed, bind to human histocompatibility leukocyte antigen (HLA) and be recognized by T cells. Here we report the fine specificity and HLA restriction of several T lymphocyte clones (TLC) specific for a peptide which is derived from the second mutational hot spot in ras encoding residue 61. These TLC were generated from memory T cells present in the blood of a cancer patient and recognized a ras-derived peptide carrying Leu instead of Gln at residue 61. By sequencing of the T cell receptor (TcR) genes three sets of "sister" TLC carrying highly different TcR were identified. Two of the TLC recognized a peptide carrying the 61 Leu mutation presented by HLA-DQ8 and one recognized the same peptide presented by HLA-DQ4. By using truncated peptides derived from residues 51 to 69 of p21 ras, partially overlapping minimal epitopes could be defined. All three TLC recognized the corresponding recombinant mutant p21 ras oncoprotein carrying Leu at residue 61 presented by autologous B-lymphoblastoid cell lines (B-LCL). This demonstrates that naturally derived ras peptides from this region of p21 ras encompass the three epitopes recognized by the TLC. These results indicate that immunogenic ras-derived peptides may be used in immunotherapy of cancer where transforming ras oncoproteins are involved.