The tumor-associated autoantigenMUC1is intensively studied as a potential target for antigen-specific immunotherapy of cancer. Previous reports concerning experiments in preclinical murine tumor models have provided evidence supporting the feasibility of this approach. However, such studies have not been performed with clonal cytotoxic T lymphocyte populations displaying a highly definedMUC1specificity. The authors demonstrate that the immunodominantMUC1-specific cytotoxic T lymphocyte response in C57BL/6 mice is directed against an H-2Kb–restricted epitope,MUC119–27, which is derived from the N-terminal signal sequence of theMUC1protein. Processing of this epitope was independent of transporter of antigen presentation and proteasome function. Importantly, successful immunotherapy ofMUC1-overexpressing tumors inMUC1-transgenic mice was not accompanied by damage to normal somaticMUC1-positive tissues, even when this involved the infusion of large numbers of clonal cytotoxic T lymphocyte that recognized the immunodominantMUC1epitope. Although the risk for autoimmune pathology is limited, data indicate that immune tolerance inMUC1-positive subjects restricts the breadth of theMUC1-specific cytotoxic T lymphocyte repertoire that is available for recruitment to immunotherapeutic antitumor responses.