Objective: Donor lymphocyte infusions may generate a desirable graft-versus-leukemia effect, but also elicit a noxious graft-versus-host disease. A positive selection of leukemia (antigen)-specific T cells would be highly desirable. In this study, we focused on the immunogenic leukemia antigen Wilms’ Tumor gene 1 (WT1). Materials and Methods: We employed the technology of streptamers available at good manufacturing practice level to first determine the frequency of human leukocyte antigen−A2 restricted WT1-specific CD8+ T cells. Then, specific cells were labeled with streptamers and selected by magnetic cell separation. Purity and immunophenotype of selected cells were analyzed. Results: Twenty-one of 40 healthy donors had naïve WT1-specific CD8+ T-cell frequencies of >0.5%, and 8 of 40 even >1.0% of all CD8+ T cells. In 7 of 10 acute myeloid leukemia patients, the frequencies were 0.5% to 3.65%. After positive selection by magnetic cell separation, a 60-fold increase with a purity of up to 17.79% in the lymphocyte gate and 86.18% in the CD8+ T-cell gate could be achieved for CD8+WT1streptamer+CD28+/−CD45RA+CCR7− effector T cells. Conclusions: Streptamer technology allows selection of pure WT1-specific effector T cells. This is a prerequisite for clinical applications targeting tumor-specific antigens, such as adoptive T-cell transfer. [ABSTRACT FROM AUTHOR]