Evidence has accumulated that the immune system can play a significant role in the defense against tumors in humans. Especially melanoma and renal cell carcinoma (RCC) are considered immunogenic tumors. In contrast to melanoma, hardly any RCC-associated antigens have been identified as targets for RCC-reactive T cells. Here, we report the identification of a human leukocyte antigen (HLA)-A2.1-restricted T-cell epitope within the G250 antigen. This antigen is expressed in 85% of RCCs but not by neighboring normal kidney tissue and has recently been molecularly defined and shown to be identical to MN/CA IX. Computer-aided motif prediction revealed the presence of 60 potential HLA-A2.1-binding peptides within the G250 antigen. Subsequent binding analysis showed that 13 of these peptides bound to HLA-A2.1 with high-to-intermediate affinity. Analysis of their immunogenicity in HLA-A2.1Kb transgenic mice indicated that 4 of the 13 peptides gave rise to cytotoxic T lymphocytes (CTLs) capable of lysing peptide-loaded target cells. However, only the G250 peptide 254-262 induced CTLs that recognized target cells that endogenously expressed the G250 antigen. Similarly, we were also able to raise human CTLs against the G250 peptide 254-262, which lysed target cells that endogenously expressed the G250 antigen. These findings and the high prevalence of this antigen in RCC patients makes G250 a potential target for anti-RCC immunotherapy.