To see whether different antigens expressed by the same tumor are recognized by distinct T ‐cell receptors (TCR), we used cytotoxic T‐lymphocyte (CTL) lines known to lyse in vitro the syngeneic BALB/c adenocarcinoma C‐26. Four of these CD3⁺ CD8⁺ lines showed 4 different patterns of lysis on a panel of MHC‐class‐I‐compatible targets. The activity was H‐2^d‐restricted and could be blocked by anti‐CD3 and anti‐TCR‐α/β monoclonal antibodies (MAbs). The CTL lines were also effective, although to a different extent, in adoptive immunotherapy of mice bearing lung metastases. Phenotypic analysis revealed in all the lines a high frequency of cells positive for CD45, asialo GMI (ASGMI), lymphocyte‐function‐associated antigen‐I (LFA‐I), intercellular adhesion molecule‐I (ICAM‐I) and CD44, but negligible expression of L‐selectin (LAM‐I) and very late antigen‐4 (VLA‐4); 2 lines expressed the vitronectin‐receptor (VNR). Analysis of TCR Vβ‐chains used by the 4 lines showed selective presence of Vβ6, Vβ8.2, Vβ8.3 and of Vβ13 chains. MAbs directed to these Vβ chains blocked their lytic activity in vitro. Vα‐chain transcripts of the lines were identified by polymerase chain reaction (PCR) as VαITTII and Vα52 in 2 lines, while one could not be identified. Analysis of Vβs in mixed lymphocyte‐tumor‐cell cultres (MLTC) of T cells deriving from tumor‐infiltrating lymphocytes (TIL) or from spleen of C‐26 tumor‐bearing or immune animals indicated that the TCR of the CTL lines were representatives of the TCR repertoire recognizing C‐26 tumor, since their Vβ were shown to be selectively expanded in MLTC. © 1994 Wiley‐Liss, Inc.