The transcription factor PAX3 is expressed during early embryogenesis and in multiple cancer types, including embryonal rhabdomyosarcoma (ERMS), Ewing sarcoma (ES) and malignant melanoma (MEL), suggesting that it could function as a general tumor associated antigen. Major histocompatibility complex (MHC) peptide binding algorithms were used to predict potential epitopes in PAX3 capable of stimulating in vitro naïve HLA-A0201 restricted cytotoxic T-lymphocytes (CTLs). Two peptides, PAX3-282 (QLMAFNHLI) and a modified version of this peptide PAX3-282.9V (QLMAFNHLV), were capable of inducing antigen-specific CTLs. Of these peptides, PAX3-282.9V was the most efficient inducer of primary CTL response. These CTLs were able to lyse HLA-A0201 expressing target cells that were pulsed with peptide, and more importantly, were effective in killing tumor cells that express PAX3, including ERMS, ES and MEL cell lines. These findings provide compelling evidence that peptide PAX3-282 is naturally processed by tumors and is presented in the context of HLA-A0201 in adequate amounts to allow CTL recognition. Also, PAX3-282.9V is an effective immunogenic peptide able to induce CTL recognition of PAX3-containing tumors and may be used as an antitumor peptide vaccine.