The WT1 gene contributes to leukemogenesis and all adult ALL, AML and CML express WT1 RNA by quantitative real-time reverse transcription polymerase chain reaction (qPCR) techniques. WT1 may therefore be a useful antigenic target for immunotherapy. Four HLA-A*0201-restricted WT1 T cell epitopes have been identified: Db126 (RMFPNAPYL), WH187 (SLGEQQYSV), WT37-45 (VLDFAPPGA) and WT235 (CMTWNQMNL), but only Db126 has been extensively studied in myeloid leukemias. Here, we sought CD8+ T cells directed against these epitopes in 12 healthy SCT donors, 6 patients with AML, 8 with, CML and 6 with ALL prior to SCT. All patients tested with myeloid or lymphoid leukemias expressed MHC class I, B7.1 and WT1. To detect very low frequencies of WT1-specific CD8+ T cells, we used qPCR for interferon-g (IFN-g) mRNA. After isolation, 106 CD8+ T cells were stimulated with C1R-A2 cells (MHC class I-defective LCL cells transfected with HLA-A*0201) loaded with test peptides at concentrations of 0.1, 1 and 10 mM to determine their functional avidity. CD8+ T cells were also stimulated with CMV pp65 (positive control) and gp100 (209-2M) (negative control) peptides. After 3 hr coculture, cells were harvested for RNA extraction and cDNA synthesis. qPCR was performed for IFN-g mRNA and normalized to copies of CD8 mRNA from the same sample. Parallel assays using tetramers demonstrated that the IFN-g copy number was linearly related to the frequency of tetramer-binding T cells, sensitive to frequencies of 1 responding CD8+ T cell/100 000 CD8+ T cells. A positive response was defined as a threshold of 100 or more IFN-g mRNA copies/104 CD8 copies and a stimulation index (SI) of 2 or more, where SI = IFN-g mRNA copies/104 CD8 copies in peptide pulsed/unpulsed cultures. Responses to at least one WT1 peptide were detected in 5/8 CML patients, 4/6 patients with AML and 7/12 healthy donors. Notably, a response was not elicited to WT1 in any of the 6 patients with ALL, despite evidence of immune competence as shown by a normal CMV response. Five of five CML responders and 3/4 AML responders recognized 2 or more WT1 epitopes, while the 7 healthy donors recognized only one WT1 epitope. The range of IFN-g mRNA copies/104 CD8 copies was 289–13584, 418–45891 and 160–2683 for CML, AML and healthy donors respectively. WT1-specific tetramer-positive CD8+ T cells displayed both central memory (CD45RO+CD27+CD57−) and terminally differentiated effector memory phenotypes (CD45RO-CD27−CD57+). As multiple WT1-derived epitopes can be targeted simultaneously, it is likely that T cell response to WT1 is polyclonal. These results are important because the presence of memory WT1 responses in patients with myeloid leukemias and healthy individuals should favor vaccination as a means to expand immune responses to leukemia in the autologous and allogeneic transplant setting. Furthermore, the presence of CD8+ T cell responses to multiple WT1 epitopes should favor robust polyclonal immune responses to leukemia. However, failure to detect CD8+ T cell responses to WT1 in ALL patients suggests that WT1 may not be a useful antigen to target for immunotherapeutic purposes in this patient group.