Tumors contain many antigens that may be recognized by the immune system. It is not known whether these antigens, and the epitopes within these antigens, can all be recognized by the anti-tumor immune response or if such responses are restricted to a few dominant epitopes. Effector function of endogenous cytotoxic T lymphocytes (CTL) generated during tumor progression has previously been assessed by indirect, ex vivo assays, which often focused on a single antigen. Therefore, we evaluated the endogenous in vivo CTL response to multiple neo tumor antigens using murine Lewis lung carcinoma tumor cells transfected with ovalbumin or a polyepitope construct. Both express multiple MHC class I-restricted epitopes. Ovalbumin contains a known hierarchy of epitopes for given MHC molecules, whilst the polyepitope expresses a number of dominant epitopes. We show that as tumors progress, potent effector CTL are generated in vivo that are restricted to dominant epitopes; we did not see the responses to subdominant or cryptic epitopes. Our data show that the CTL recognizing tumor antigens vary in their lytic capacity, as the CTL responding to two of the four epitopes were particularly potent killers. The presence of these effector CTLs did not prevent tumor growth. However, intra-tumoral IL-2 treatment altered the potency, but not the hierarchy, of these CTL such that they mediated tumor regression. These results have implications for immunotherapy protocols.