Immunotherapeutic strategies targeting the rare leukemic stem cell compartment might provide salvage to the high relapse rates currently observed in acute myeloid leukemia. We applied gene expression profiling for comparison of leukemic blasts and leukemic stem cells with their normal counterparts. Here, we show that the T-cell receptor γ chain alternate reading frame protein (TARP) is overexpressed in de novo pediatric (n=13) and adult (n=17) acute myeloid leukemia sorted leukemic stem cells and blasts compared to hematopoietic stem cells and normal myeloblasts (15 healthy controls). Moreover, TARP expression was significantly associated with a fms-like tyrosine kinase receptor-3 internal tandem duplications in pediatric acute myeloid leukemia. TARP overexpression was confirmed in acute myeloid leukemia cell lines (n=9), and was found to be absent in B-cell acute lymphocytic leukemia (n=5) and chronic myeloid leukemia (n=1). Sequencing revealed that both a classical TARP transcript, as described in breast and prostate adenocarcinoma, and an acute myeloid leukemia-specific alternative TARP transcript, were present. Protein expression levels mostly matched transcript levels. TARP was shown to reside in the cytoplasmic compartment and showed sporadic endoplasmatic reticulum co-localization. TARP-T-cell receptor engineered cytotoxic T-cells in vitro killed acute myeloid leukemia cell lines and patient leukemic cells co-expressing TARP and HLA-A*0201. In conclusion, TARP qualifies as a relevant target for immunotherapeutic T-cell therapy in acute myeloid leukemia.