Restricted and high-level expression of interleukin-13 receptor α2 (IL-13Rα2) in a majority of human malignant gliomas makes this protein an attractive vaccine target. We have previously described the identification of the IL-13Rα2_345-353 peptide as a human leukocyte antigen-A2 (HLA-A2)–restricted CTL epitope. However, as it remains unclear how efficiently peptide-based vaccines can induce specific CTLs in patients with malignant gliomas, we have examined whether analogue epitopes could elicit heteroclitic antitumor T-cell responses versus wild-type peptides. We have created three IL-13Rα2 analogue peptides by substitutions of the COOH-terminal isoleucine (I) for valine (V) and the NH₂-terminal tryptophan (W) for either alanine (A), glutamic acid (E), or nonsubstituted (W; designated as 1A9V, 1E9V, and 9V, respectively). In comparison with the native IL-13Rα2 epitope, the analogue peptides 9V and 1A9V displayed higher levels of binding affinity and stability in HLA-A2 complexes and yielded an improved stimulatory index for patient-derived, specific CTLs against the native epitope expressed by HLA-A2⁺ glioma cells. In HLA-A2-transgenic HHD mice, immunization with the peptides 9V and 1A9V induced enhanced levels of CTL reactivity and protective immunity against an intracranial challenge with IL13Rα2-expressing syngeneic tumors when compared with vaccines containing the native IL-13Rα2 epitope. These findings indicate highly immunogenic IL-13Rα2 peptide analogues may be useful for the development of vaccines capable of effectively expanding IL-13Rα2-specific, tumor-reactive CTLs in glioma patients. (Cancer Res 2006; 66(11): 5883-91)