bThe DtxR family consists of metal-dependent transcription factors (DtxR-TFs) that regulate the expression of genes involved in metal homeostasis in the cell. The majority of characterized DtxR-TFs belong to Bacteria. In the current work, we applied a comparative genomics approach to predict DNA-binding sites and reconstruct regulons for DtxR-TFs in Archaea. As a result, we inferred 575 candidate binding sites for 139 DtxR-TFs in 77 genomes from 15 taxonomic orders. Novel DNA motifs of archaeal DtxR-TFs that have a common palindromic structure were classified into 10 distinct groups. By combining functional regulon reconstructions with phylogenetic analysis, we selected 28 DtxR-TF clades and assigned them metal specificities and regulator names. The reconstructed FetR (ferrous iron), MntR (manganese), and ZntR (zinc) regulons largely contain known or putative metal uptake transporters from the FeoAB, NRAMP, ZIP, and TroA families. A novel family of putative iron transporters (named Irt), including multiple FetR-regulated paralogs, was identified in iron-oxidizing Archaea from the Sulfolobales order. The reconstructed DtxR-TF regulons were reconciled with available transcriptomics data in Archaeoglobus, Halobacterium, and Thermococcus spp.T ransition metals, including iron, manganese, and zinc, are responsible for a diverse array of biochemical reactions and other biological functions in prokaryotes. The particular properties of ferrous iron (Fe 2ϩ ) and ferric iron (Fe 3ϩ ) ions make them widely used redox-sensing elements in many metalloenzymes, from heme-containing cytochromes to Fe-S proteins. Manganese is used in free radical-detoxifying enzymes, including superoxide dismutase and catalase and some other enzymes. Zinc was found in many proteins, including enzymes of nucleic acid metabolism and ribosomal proteins, where it plays a role in both catalysis and protein structure. Many transition metals are taken up by specific transport systems, including FeoAB family transporters for ferrous iron (1, 2), Fbp-type ATP-binding cassette (ABC) transporters for ferric iron (3), Znu family ABC transporters for zinc (4), and NRAMP family MntR transporters for manganese (5). The precise maintenance of metal ion homeostasis, including metal uptake transporters, is vital for cells that have to balance between supplying enzymes with metal ion cofactors and decreasing the harmful effects of heavy metals (6, 7).The regulation of gene expression by specific binding of transcription factors (TFs) to their DNA sites in response to a cellular signal (e.g., specific metal ions) is a common regulatory mechanism in microorganisms. Iron, manganese, and zinc homeostasis genes in prokaryotes are controlled by TFs from two major families of metalloregulators, Fur and DtxR (8). The ferric uptake regulator Fur and zinc uptake regulator Zur constitute the majority of the Fur family metalloregulators, being widely distributed in diverse lineages of Bacteria but not Archaea, whereas the manganese-and nickel-specific regulators Mur and Nur...