Comparative genomic analysis was applied to identify the biotin transcriptional regulator, BioR, in most Alphaproteobacteria, and to identify its recognition signal TTATMKATAA. BioR belongs to the GntR family of transcriptional repressors. The functional assignment is supported by three lines of evidence: (1) bioR is positionally clustered with various bio genes, both for biotin biosynthesis and transport; (2) in most cases, candidate BioR-binding sites (BIOR boxes) are observed upstream of the bioR genes, suggesting autoregulation; (3) the phyletic distribution of the BIOR boxes coincides exactly with the phyletic distribution of the bioR genes, as the genomes lacking BIOR boxes do not have orthologs of bioR. Thus, in Alphaproteobacteria, BioR seems to have assumed the role of the biotin regulator that in most other bacteria is fulfilled by the dual function biotin-protein ligase BirA having the DNA-binding helix-turn-helix domain.