The RHAMM/CD168-derived peptide R3 is naturally processed. R3-primed effector cells showed specific lysis of RHAMM/CD168&HLA-A2–transfected COS7 cells. COS7 cells were transfected with the plasmid vector pBudCE4.1 containing RHAMM/CD168 and/or HLA-A2. T lymphocytes generated in an MLPC with the RHAMM/CD168-derived peptide R3 were able to lyse COS7 cells double transfected with HLA-A2 and RHAMM/CD168 but not mock-transfected or solely HLA-A2– or RHAMM/CD168-transfected cells (A). Addition of an anti–HLA-ABC antibody but not an IgG isotype antibody (ISO) abolished the lysis of T2 pulsed with the R3 peptide by R3-primed T lymphocytes (B). K562 cells lacking HLA-ABC molecules but expressing RHAMM/CD168 could not be lysed by R3-pulsed T cells (B).