Acetylation of histone core particles plays an important role in modulating chromatin structure and gene expression. The acetylation status of the histone tails is determined by two opposing enzymatic activities, histone acetyltransferases and histone deacetylases (HDACs). Here we describe the isolation and characterization of HDAC10, a novel class II histone deacetylase. Molecular cloning and Northern blot analyses reveal that the HDAC10 transcript is widely expressed and subjected to alternative splicing. HDAC10 is both nuclear and cytoplasmic, a feature reminiscent of HDACs 4, 5, and 7. Distinct from other family members, HDAC10 harbors an amino-terminal catalytic domain and a carboxyl pseudo-repeat that shares significant homology with its catalytic domain. Mutational analysis reveals that transcriptional repression by HDAC10 requires its intrinsic histone deacetylase activity. Taken together, HDAC10 represents a distinct HDAC that may play a role in transcription regulation.