The retrovirus-induced RBL5 lymphoma can be rejected by adoptive transfer of noncytolytic CD4+ Th 1 lymphocytes in normal hosts, without a requirement for transfer of specific CD8+ CTL. Therefore, we hypothesized that host precursor CTL (pCTL) might cooperate with transferred CD4+ Th1 cells to mediate tumor rejection. To evaluate this hypothesis, lymphocytes from non-immunized mice were analyzed for cytolytic activity after short-term bulk lymphocyte tumor culture (BLTC) with rIL-2 (5U/ml). BLTC induced the differentiation of anti-RBL5 CTL distinct from non-MHC-restricted LAK. These effectors were CD8+, TCR alpha/beta+, and utilized the CD3-TCR complex for MHC class I-restricted lysis. The majority of pCTL were found within the CD44/PgP-1hi population of memory/activated lymphocytes. However, there was no serologic evidence for prior exposure to RBL5-related tumor or viral Ags. CTL activity was susceptible to partial blockade with mAbs directed against CD8 and MHC class I, suggesting a relatively low-affinity Ag-TCR interaction. These data are most consistent with the recruitment of a population of Ag-specific, but cross-reactive, pCTL during BLTC.