Zinc-dependent histone deacetylases (HDACs) are a family of hydrolases first identified as components of transcriptional repressor complexes, where they act by deacetylating lysine residues at the N-terminal extensions of core histones thereby affecting transcription. To get more insight into the biological functions of the individual HDAC family members we have used RNAi in combination with microarray analysis in Drosophila S2 cells. Silencing of Drosophila HDAC1 (DHDAC1) but not of the other DHDAC family members lead to increased histone acetylation. Silencing of either DHDAC1 or DHDAC3 lead to cell growth inhibition and deregulated transcription of both common and distinct groups of genes. Silencing of DHDAC2 lead to increased tubulin acetylation levels but was not associated with a deregulation of gene expression. No growth phenotype and no significant deregulation of gene expression was observed upon silencing of DHDACs 4 and X. Loss of DHDAC1 or exposure of S2 cells to the small molecule HDAC inhibitor Trichostatin both lead to a G2 arrest and were associated with significantly overlapping gene expression signatures in which genes involved in nucleobase and lipid metabolism, DNA replication, cell cycle regulation and signal transduction were over-represented. A large number of these genes were shown to be also deregulated upon loss of the co-repressor SIN3 (J. Biol. Chem. 278, 3780, 2003). We conclude that: 1. DHDACs 1 and 3 have distinct functions in the control of gene expression; 2.Under the tested conditions, DHDACs 2, 4 and X have no detectable transcriptional functions in S2 cells; 3. The antiproliferative and transcriptional effects of Trichostatin are largely recapitulated by the loss of DHDAC1. 4. The deacetylase activity of DHDAC1 significantly contributes to the repressor function of SIN3.