The tumor antigen PRAME (preferentially expressed antigen of melanoma) is expressed in up to 50% of patients with AML. Four HLA-A*0201-restricted epitopes in the PRAME protein have been identified: P100-108 (P100), P142-151 (P142), P300-309 (P300) and P425-433 (P425). To detect very low frequencies of PRAME-specific CD8+ T cells, we used quantitative real-time reverse transcription polymerase chain reaction (qPCR) to measure interferon-g mRNA (IFN-g) production by PRAME peptide pulsed CD8+ T cells from 11 healthy donors and 10 HLA-A*0201+ patients with AML, one of whom had received an allogeneic stem cell transplant (SCT). After isolation, 1 x106 CD8+ T cells were stimulated in vitro with C1R-A2 cells (an MHC class I-defective LCL expressing HLA-A*0201) loaded with test peptides at concentrations of 0.1, 1 and 10 mM, to determine functional avidity. CD8+ T cells were also stimulated with CMV pp65 (positive control) and gp100 (209-2M) (negative control) peptides. After 3h 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 the IFN-g copy number to be 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 cultures/unpulsed cultures. Using this sensitive technique, we found responses in 8/11 HLA-A2-positive healthy donors and 7/10 AML patients. Four of eight healthy donors and 5/7 AML responders recognized 2 or 3 peptide epitopes. The mean response against each of the four epitopes was greater in leukemic patients compared with healthy donors (3597–9371 versus 172–1288 IFN-g mRNA copies/104 CD8 copies). PRAME peptide mediated responses, particularly to P300, were also detected using an optimized ELISPOT assay in 2/4 AML patients and 5/8 normal donors tested. In cross-comparison of 8 qPCR positive donors, 6 also generated IFN-g ELISPOTS confirming IFN-g mRNA transcription was also associated with protein secretion. Of note, the most immunogenic epitope in both donors and patients by both methods was P300. Six qPCR and ELISPOT assays were concordant, but there were 2 ELISPOT negative, qPCR positive patients, and 1 ELISPOT positive qPCR negative patient. In 2/5 samples tested (1 donor and 1 patient), peptide-specific ELISPOT responses expanded from 99 to 1627 and 280 to 758 spots per million plated PBMC respectively, after a single 7 day peptide pulse. Samples from a stem cell donor and the recipient pre and post SCT were also studied. The donor had CD8+ T-cell reactivity to P142, P300 and P425. The patient had no PRAME response prior to SCT but after SCT developed significant responses to P142 and P425 and P100, suggesting the transfer and expansion of PRAME-specific CD8+ T cells from donor to recipient. These results provide the first evidence for spontaneous T-cell reactivity against PRAME in healthy donors and AML patients. They support the immunogenicity of PRAME and its potential application in immunotherapy of leukemia.