Identification of cytolytic T lymphocyte (CTL) epitopes presented by major histocompatibility complex (MHC) class I molecules on tumor cells is critical for the design of active immunotherapy. We describe the use of combinatorial peptide libraries with defined amino acids in two MHC anchor positions to search for epitopes that are recognized by H-2Db- and Kb-restricted CTL specific for the mouse lymphoma EL4. An iterative strategy was used for screening libraries in which 16 amino acids were divided into 3 groups and 3 subgroups: alpha (AL, VT, FY); beta (GS, P, DE); gamma (KR, H, NQ). The proportions of each group and subgroup at individual peptide positions were changed in the library synthesis, and the effect of these changes on CTL activity was measured in a sensitive RMA-S cell assay. A single H-2Db epitope mimic was deduced from the original library that contained > 2 x 10(8) potential peptides and was at least 9 logs more potent than the original library. Immunization of syngeneic mice with this peptide elicited CTL that lysed EL4 cells as well as RMA-S cells pulsed with peptides isolated from Db molecules of EL4 cells, indicating functional similarity between the mimicking peptide and the naturally processed CTL epitope. Furthermore, adoptive transfer of such a CTL line had a therapeutic effect in mice with EL4 established as an ascites tumor. Two H-2Kb-restricted epitope mimics of the same tumor were also identified. Our method represents a novel approach for the construction of MHC class I-restricted targets that can serve as immunogens for active immunotherapy of cancer.