The steady-state level of histone acetylation in eukaryotes is established and maintained by multiple histone acetyltransferases (HATs) and histone deacetylases (HDACs) and affects both the structure and the function of chromatin. Histone deacetylases play a key role in the regulation of transcription, and form a highly conserved protein family in many eukaryotic species. Here we describe the cloning, sequencing and genetic mapping of two histone deacetylase genes in Drosophila melanogaster: dHDAC1 is essentially identical to the previously cloned D. melanogaster d-Rpd3 gene and dHDAC3, a novel gene, is orthologous to the human and the chicken (Gallus gallus) HDAC3 genes. The predicted amino acid sequence (438 aa) of dHDAC3 shows 58.1% identity with dHDAC1/d-Rpd3, the only previously known member of the HDAC family in this organism. The map positions on polytene chromosomes for dHDAC1 and dHDAC3 were determined as 64C1-6 and 83A3-4 respectively. A search for other dHDAC3-like genes failed to find other potential paralogues in D. melanogaster, but identified significant homologies with bacterial and fungal genes encoding enzymes that metabolise acetyl groups, and with genes for other hydrolyases such as carboxypeptidase. In addition, histone deacetylase activity in D. melanogaster nuclear extracts can be inhibited by high concentrations of zinc and activated by low concentrations, which is identical to the properties of bovine carboxypeptidase A. On the basis of sequence and functional similarities, we suggest that histone deacetylases are metal-substituted enzymes.