Histone deacetylases (HDACs) are a family of enzymes involved in the regulation of gene expression, DNA repair, and stress response. These processes often are altered in tumors, and HDAC inhibitors have had pronounced antitumor activity with promising results in clinical trials. Here, we report the crystal structure of human HDAC8 in complex with a hydroxamic acid inhibitor. Such a structure of a eukaryotic zinc-dependent HDAC has not be described previously. Similar to bacterial HDAC-like protein, HDAC8 folds in a single ␣͞ domain. The inhibitor and the zinc-binding sites are similar in both proteins. However, significant differences are observed in the length and structure of the loops surrounding the active site, including the presence of two potassium ions in HDAC8 structure, one of which interacts with key catalytic residues. CD data suggest a direct role of potassium in the fold stabilization of HDAC8. Knockdown of HDAC8 by RNA interference inhibits growth of human lung, colon, and cervical cancer cell lines, highlighting the importance of this HDAC subtype for tumor cell proliferation. Our findings open the way for the design and development of selective inhibitors of HDAC8 as possible antitumor agents.T he epigenetic control of gene expression is operated through a series of posttranslational modifications of chromatin that influence the electrostatics of DNA-protein interactions and generate docking sites for a large number of chromatininteracting proteins (1, 2). The acetylation status of lysine residues found in the accessible N termini of core histones is one of the posttranslational chromatin modifications that impinge on gene expression. Acetylation and deacetylation of histones are controlled by the enzymatic activity of histone acetyltransferases and histone deacetylases (HDACs) (3, 4). Alterations of gene expression are a hallmark of cancer, and mounting evidence suggests that at least a part of these alterations is mediated by epigenetic mechanisms (5, 6). Importantly, the aberrant recruitment of HDACs has been mechanistically linked to malignancy in leukemias and lymphomas (7,8), and small-molecule HDAC inhibitors show antitumor activity in preclinical models and in clinical trials and have the promise to become effective, new antineoplastic therapeutics (9).At least 18 HDAC subtypes exist, and they are subdivided into three classes (10): class I (HDACs 1-3 and 8), homologous to the yeast Rpd3 deacetylase; class II (HDACs 4-7, 9, and 10), related to the yeast Hda1 deacetylase; and class III proteins (Sirtuins 1-7), which are yeast Sir2 homologs. HDAC11 has homology to both class I and II enzymes but cannot unambiguously be assigned to either class. Class I and II HDACs, as well as HDAC11, are all zinc-dependent hydrolases. The therapeutically relevant HDAC inhibitors are thought to be nonselective or poorly selective inhibitors of all or most of class I and II enzymes but do not inhibit class III HDACs (9). It is not clear whether the antitumor properties of HDAC inhibitors are due to their l...