Tumor infiltrating lymphocytes (TIL) were cultured from multiple methylcholanthrene-induced sarcomas under four different conditions: either low-dose (10 U/ml) or high-dose (1000 U/ml) rIL-2 was used with Ag stimulation by irradiated autologous tumor and splenocytes starting either at day 1 or day 10 of culture. TIL grown from four antigenically distinct sarcomas in low-dose rIL-2 were specifically lytic in vitro to their tumor of origin in 13 of 15 (87%) lytic cultures whereas only 8 of 28 (29%) lytic TIL cultures grown in high-dose rIL-2 showed specificity. TIL cultured in low-dose rIL-2 with Ag stimulation on day 1 of culture proliferated at a rate equal to TIL grown in high-dose rIL-2 and maintained their specificity for over 3 mo in culture. Cytolysis by specific TIL was MHC-restricted. TIL with in vitro specificity were therapeutically effective in eliminating established micrometastases in murine models. These investigations demonstrate that CTL derived from tumor-bearing mice can be used to define unique tumor-associated Ag on at least four different sarcomas and may be valuable in studies of the biologic nature of these Ag and in the adoptive immunotherapy of tumors.