Abstract Purpose: This study aimed to examine CD8 T-cell reactivity in breast cancer patients against cyclin B1–derived peptides restricted by the human leukocyte antigen (HLA)-A2 molecule. Experimental Design: Peripheral blood mononuclear cells from 36 breast cancer patients were analyzed by enzyme-linked immunosorbent spot (ELISPOT) for the presence of T cells recognizing the cyclin B1–derived peptides CB9 (AKYLMELTM) and CB-P4 (AKYLMELCC), in addition to modified versions of CB9, CB9L2 (ALYLMELTM) and CB9M2 (AMYLMELTM), both of which display higher affinity to HLA-A2. Results: Twelve patients harbored a memory CD8 T-cell response against at least one of the peptides; strongest reactivity was detected against the CB9L2 peptide. Because the level of cyclin B1 has been shown to be influenced by the level of p53, which in turn is elevated in cancer cells because of point mutation, we analyzed the level of p53 protein in biopsies from the patients by immune histochemistry. Combined data showed that anti–cyclin B1 reactivity was predominantly detected in patients with tumors characterized by elevated expression of p53. Interestingly, no reactivity was detected against six peptides derived from the p53 protein. Conclusions: Our data support the notion of cyclin B1 as a prominent target for immunologic recognition in cancer patients harboring p53-mutated cancer cells. Because mutation of p53 is one of the most frequent genetic alterations in human cancers, this suggests that immunotherapy based on targeting of cyclin B1 is broadly applicable in a large proportion of cancer patients.