In order to determine whether lymphocytes with therapeutic potential can be obtained from colon carcinoma (C26)-bearing (TB) mice, splenocytes were activated either in mixed lymphocyte-tumor cell (MLTC) cultures in the presence of 5 and 10 U/ml IL2 or 250 and 100 U/ml IL2 (LAK effectors). The therapeutic efficacy, as well as functional and phenotypic features of such lymphocytes, was then compared in an adjuvant immunotherapy setting. Comparisons of the antigenic phenotype, cytotoxic and proliferative activities, and of transcription of different cytokine genes (IFN gamma, TNF alpha, IL4, IL6) between lymphocytes activated in MLTC and LAK failed to reveal major differences. However, the in vitro lysis of C26 by MLTC-activated but not by LAK TB lymphocytes was significantly blocked by anti-T3 and anti-Lyt2 monoclonal antibodies, suggesting that a fraction of specific antitumor effectors was present in the MLTC bulk population. Moreover, the amount of IFN gamma (but not of other cytokines) produced by MLTC-derived lymphocytes after stimulation with C26 cells was shown to be 10-fold higher than that produced by LAKs. When combined with low-dose IL2 administration as an adjuvant treatment in C26-operated mice, MLTC effectors showed a higher therapeutic activity than LAKs obtained from the same pool of lymphocytes from TB donors. In the same setting, MLTC-activated lymphocytes obtained from TB or tumor-excised (TE) mice, combined with IL2, were equally effective (76 and 74% survivors, respectively, vs. 27% of the surgery control group and 26% of the group given IL2 only), whereas LAK cells from TE but not from TB animals resulted in the cure of a significant fraction of mice.