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-2drestricted and could be blocked by antLCD3 and anti-TCR-dP 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 GM I (ASGM I), 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 (VN-R). Analysis of TCR VP-chains used by the 4 lines showed selective presence of Vp6, Vp8.2, Vp8.3 and of Vp13 chains. MAbs directed to these Vf3 chains blocked their lytic activity in vitro. Va-chain transcripts of the lines were identified by polymerase chain reaction (PCR) as VolllTl I and Va52 in 2 lines, while one could not be identified. Analysis of Vps in mixed lymphocyte-tumor-cell cultures (MLTC) of T cells deriving from tumor-infiltrating lymphocytes (TlL) 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 Vps were shown to be selectively expanded in MLTC.