We previously showed (E. Clave et al., J. Immunother., 22: 1–6, 1999; J. Molldrem et al., Blood, 88: 2450–2457, 1996) that PR1, a human-lymphocyte-antigen (HLA)-A2.1-restricted peptide from proteinase 3, could be used to elicit CTLs from normal individuals. These CTLs showed HLA-restricted cytotoxicity and colony inhibition of myeloid leukemia cells that overexpress proteinase 3. In this study, we constructed a phycoerythrin-labeled PR1-HLA-A2 tetramer to identify PR1-specific CTLs by flow cytometry. No peripheral blood lymphocytes from three HLA-2.1⁺ donors stained with the tetramer, but, after 20 days in culture with weekly PR1 stimulation, 2–8% became tetramer⁺. Tetramer staining identified up to 40-fold more PR1-specific CTLs than were identified by limiting dilution analysis and correlated better with lysis of PR1-coated T2 cells (R² = 0.95 versus R² = 0.76). Tetramer⁺ CTLs were memory phenotype (91% CD45RO⁺), and most (58% CD95⁺) were activated. Tetramer-sorted allogeneic CTLs produced 83% lysis of HLA-A2.1⁺ chronic myelogenous leukemia (CML) blasts at an E:T ratio of 2.5:1, compared with 23% lysis by nonsorted CTLs, with no background lysis of HLA-A2.1⁺ normal cells. Cytoplasmic proteinase-3 expression was one log greater in CML blasts than in normal granulocytes. These results show that a PR1-HLA-A2 tetramer can be used to identify and select CTLs from normal donors that preferentially lyse CML cells, which could be used for leukemia-specific adoptive immunotherapy.