Targeted T-cell therapy is a potentially less toxic strategy than allogeneic stem cell transplantation for providing a cytotoxic antileukemic response to eliminate leukemic stem cells (LSCs) in acute myeloid leukemia (AML). However, this strategy requires identification of leukemia-associated antigens that are immunogenic and exhibit selective high expression in AML LSCs. Using microarray expression analysis of LSCs, hematopoietic cell subpopulations, and peripheral tissues to screen for candidate antigens, cyclin-A1 was identified as a candidate gene. Cyclin-A1 promotes cell proliferation and survival, has been shown to be leukemogenic in mice, is detected in LSCs of more than 50% of AML patients, and is minimally expressed in normal tissues with exception of testis. Using dendritic cells pulsed with a cyclin-A1 peptide library, we generated T cells against several cyclin-A1 oligopeptides. Two HLA A*0201-restricted epitopes were further characterized, and specific CD8 T-cell clones recognized both peptide-pulsed target cells and the HLA A*0201-positive AML line THP-1, which expresses cyclin-A1. Furthermore, cyclin-A1–specific CD8 T cells lysed primary AML cells. Thus, cyclin-A1 is the first prototypic leukemia-testis-antigen to be expressed in AML LSCs. The pro-oncogenic activity, high expression levels, and multitude of immunogenic epitopes make it a viable target for pursuing T cell–based therapy approaches.