Identification of tumor-associated antigens and advances in tumor immunology resulted in the development of vaccination strategies to treat patients with malignant diseases. Using a novel approach that combines DNA chip analysis of tumor samples with isolation of peptides on the surface of tumor cells, a HLA-A*0201-binding peptide derived from the adipophilin protein was identified. Adipophilin is involved in lipid storage and was thought to be expressed only in adipocytes, but it can be found in other cell types such as macrophages or tumor cells. In the present study, we analyzed the possible use of this peptide as a T-cell epitope presented by malignant cells. To accomplish this, we induced CTL responses using this HLA-A*0201-binding peptide. The in vitro-induced CTLs efficiently lysed cells pulsed with the adipophilin peptide and HLA-matched tumor cell lines in an antigen-specific and HLA-restricted manner. Finally, the induced CTLs recognized autologous dendritic cells (DCs) pulsed with the antigenic peptide or transfected with tumor RNA purified from an adipophilin-expressing tumor cell line. To further analyze the possible use of this peptide in immunotherapies of human malignancies, we induced adipophilin-specific CTLs using peripheral blood mononuclear cells and DCs from HLA-A*0201-positive patients with chronic lymphatic leukemia and plasma cell leukemia. The in vitro-generated CTLs recognized autologous chronic lymphatic leukemia cells and malignant plasma cells, whereas they spared nonmalignant resting or activated B and T lymphocytes, monocytes, or DCs. Our results demonstrate that this peptide might represent an interesting candidate for the development of cancer vaccines designed to target adipophilin-derived epitopes in a wide range of malignancies.