Material eluted from RMA lymphoma or B6 spleen cells under acid conditions was fractionated by reverse phase high-performance liquid chromatography, and tested for ability to restore the sensitivity to cytotoxic T lymphocytes of the processing/presentation defective mutant line RMA-S. This allowed identification of three fractions (termed M1, M2 and M3) carrying B6 antigens recognized by cytotoxic T lymphocytes (CTL) elicited across the minor histocompatibility barrier A.BY anti-B6 (both H-2b) and one fraction (termed T1) carrying a tumor antigen recognized by B6 anti-RMA CTL. By parallel runs of material from cell lysates over major histocompatibility complex class I affinity columns, the M2 and M3 antigens were defined as Kb restricted, and M1 and T1 as Db restricted. Isolated fractions loaded onto RMA-S cells could be used to prime anti-minor histocompatibility antigen and tumor CTL in vivo. They could also be used for in vitro restimulation of spleen cells from mice that had been primed either by antigen-loaded RMA-S, or by wild-type RMA tumor cells and B6 splenocytes. The CTL generated by these methods were specific for the loading antigen, and they also recognized the antigen on the "physiological" target, i.e. RMA or B6 lymphoblasts. This system based on RMA-S as an immunization and target antigen reporter cell may be used for dissection of complex CTL responses, e.g. in studies of clonal composition and epitope dominance, or for studies of tumors that are poor stimulators of immunity.