Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes found in prokaryotes and eukaryotes. NATs have been characterized in bacteria (Bacilli, Mycobacteria, Salmonella etc.), laboratory animals (chicken, rabbit, rodents etc.) and humans, where the NAT loci occupy 230 kilobases on chromosome 8p22. Our previous comprehensive search for NAT genes involved 416 genomes (340 prokaryotic, 76 eukaryotic) and identified NAT homologues in several taxa, while also reporting on taxa that appeared to lack NAT genes [Boukouvala, S. and Fakis, G. (2005) Drug Metab. Rev. 37(3), 511-564]. Here, we present an update of this genomic search, covering 2138 genomes (1674 prokaryotic, 464 eukaryotic), of which 1167 (986 prokaryotic, 181 eukaryotic) were accessible using the advanced search algorithm tBLASTn. We have reconstructed the full-length open reading frames for putative proteins with sequence homology and features characteristic of NAT from 274 bacterial genomes (31 actinobacteria, 6 bacteroidetes/chlorobi, 2 cyanobacteria, 65 firmicutes and 170 proteobacteria) and 27 animals (1 sea-urchin, 5 fishes, 1 lizard, 1 bird and 19 mammals). Partial NAT sequences were recovered from several other organisms, including fungi, where NAT genes were found in 30 ascomycetes and 2 basidiomycetes. No NATs were found in arhaea, plants and lower invertebrates (insects and worms), while it is also uncertain whether NAT genes exist in protista. We present comparative genomic and phylogenetic analyses of the identified NAT homologues and announce a new database that will maintain information on non-human NATs and will provide recommendations for a standardized nomenclature, along the lines of the NAT Gene Nomenclature Committee.