BACKGROUND: Ependymoma are primary tumors of the nervous system. Due to their growth pattern, many ependymomas can be managed with neurosurgical resection alone. A substantial proportion of these tumors recurs or displays infiltrative growth patterns. Further established therapeutic options include radiation therapy. Systemic treatment options include platinum-based therapeutic regimes or a combination of lapatinib and temozolomide. Peptide-based immunotherapy represents a promising therapeutic strategy relying on the induction of tumor-specific T cells targeting human leukocyte antigens (HLA)-presented peptides. Our work aimed to analyze the landscape of naturally presented HLA class I and II ligands of primary ependymomas (EPN) to delineate EPN-associated antigens. METHODS: We investigated 22 EPN tissue samples using a comparative mass spectrometry-based immunopeptidomic approach. Additionally, EPN-specific antigens were functionally characterized in T-cell-based immunogenicity assays. RESULTS: We discovered a subset of EPN-exclusive peptides including HLA-A02 and HLA-A25/HLA-A26-restricted HLA ligands and identified a small panel of cancer/testis antigens (CTAs)-derived HLA ligands. Furthermore, we outlined immunopeptidomic alterations in different ependymoma subgroups and progressive ependymoma. Subsequently, we performed functional characterization of the previously identified HLA-A02:01 restricted peptide FLDS to demonstrate immunogenicity in vitro. CONCLUSION: The immunopeptidome landscape of EPNs provides actionable targets that could further be explored as a T cell-based immunotherapeutic strategy in this tumor entity.