EphA2 is a receptor tyrosine kinase, is frequently overexpressed in a wide array of advanced cancers. We demonstrate in the current study that the EphA2 protein is restrictedly expressed in primary glioblastoma multiforme, anaplastic astrocytoma tissues in comparison to normal brain tissues. To evaluate the possibility of targeting EphA2 in glioma vaccine strategies, we stimulated human leukocyte antigen (HLA) A2⁺ peripheral blood mononuclear cells (PBMCs) obtained from healthy donors, glioma patients with autologous dendritic cells (DCs) loaded with synthetic EphA2$$3_$91 peptide (TLADFDPRV), which has previously been reported to induce interferon-δ in HLAA2⁺ PBMCs. Stimulated PBMCs demonstrated antigenspecific cytotoxic T lymphocyte (CTL) responses as detected by specific lysis of T2 cells loaded with the EphA2₈₈₃ peptide as well as HLA-A2⁺ glioma cells, SNB19, U251, that express EphA2. Furthermore, in vivo immunization of HLA-A2 transgenic HHD mice with the EphA2_883-891 peptide resulted in the development of an epitope-specific CTL response in splenocytes, despite the fact that EphA2_883-891 is an autoantigen in these mice. Taken together, these data suggest that EphA2_883-891 may be an attractive antigen epitope for molecularly targeted glioma vaccines.