Lysine residues on the N-terminal tails of histones in chromatin are the primary targets of histone acetyltransferases (HATs) and histone deacetylases (HDACs) in eukaryotes. Regulation of histone acetylation by these two classes of enzymes plays significant roles in controlling transcriptional activity in cells. Eukaryotic organisms have several different HDACs, but the biological roles of each HDAC are still not clear. To understand the physiological functions of HDACs, we characterized six different Drosophila HDACs, including Rpd3, HDAC3, HDAC4, HDAC6-S, HDAC6-L, and Sir2, by developmental expression pattern, transcriptional profiles of target genes, and sensitivity to HDAC inhibitors. We found that each HDAC has a distinct temporal expression pattern and regulates transcription of a unique set of genes. Furthermore, we demonstrated differential sensitivity of HDACs to inhibitors. These results show that each individual HDAC plays different roles in regulating genes involved in various biological processes.