Allogeneic hematopoietic stem cell transplantation can induce considerable tumor remissions in metastatic renal-cell carcinoma (RCC) patients. The precise effector mechanisms mediating these graft-versus-tumor reactions are unknown. We studied RCC-directed CD8(+) T-cell responses in blood lymphocytes of healthy individuals matched with established RCC cell lines for HLA-class I. In 21 of 22 allogeneic mixed lymphocyte/tumor-cell cultures (MLTCs), RCC-reactive cytotoxic T-lymphocytes (CTLs) were readily obtained. From MLTCs, 121 CD8(+) CTL clones with memory phenotype were isolated. Their anti-RCC reactivity was restricted by multiple classical HLA-Ia molecules, in particular by HLA-A2, -A3, -B7, -B44, -Cw7, and by a nonclassical HLA-Ib determinant. Extensive cross-reactivity analyses on a broad target panel identified CTLs that recognize antigens with expression restricted to renal tissue or to renal and colon tumors. Other CTLs were directed against antigens with broader tissue distribution being expressed in various epithelial and nonepithelial tumors or, additionally, in hematopoietic cells. With microcapillary liquid chromatography and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)/TOF mass spectrometry, we identified the HLA-A*0301-associated nonpolymorphic peptide KLPNSVLGR encoded by the ubiquitously expressed Eps15 homology domain-containing 2 gene as a CTL target. Defining human RCC antigens recognized by alloreactive CTLs may allow to improve the specificity and efficiency of allogeneic cell therapy (eg, specific donor-lymphocyte infusions or vaccination) in metastatic RCC patients.