Biotin is a necessary cofactor of numerous biotin-dependent carboxylases in a variety of microorganisms. The strict control of biotin biosynthesis in Escherichia coli is mediated by the bifunctional BirA protein, which acts both as a biotin-protein ligase and as a transcriptional repressor of the biotin operon. Little is known about regulation of biotin biosynthesis in other bacteria. Using comparative genomics and phylogenetic analysis, we describe the biotin biosynthetic pathway and the BirA regulon in most available bacterial genomes. Existence of an N-terminal DNA-binding domain in BirA strictly correlates with the presence of putative BirA-binding sites upstream of biotin operons. The predicted BirA-binding sites are well conserved among various eubacterial and archaeal genomes. The possible role of the hypothetical genes bioY and yhfS-yhfT, newly identified members of the BirA regulon, in the biotin metabolism is discussed. Based on analysis of co-occurrence of the biotin biosynthetic genes and bioY in complete genomes, we predict involvement of the transmembrane protein BioY in biotin transport. Various nonorthologous substitutes of the bioC-coupled gene bioH from E. coli, observed in several genomes, possibly represent the existence of different pathways for pimeloyl-CoA biosynthesis. Another interesting result of analysis of operon structures and BirA sites is that some biotin-dependent carboxylases from Rhodobacter capsulatus, actinomycetes, and archaea are possibly coregulated with BirA. BirA is the first example of a transcriptional regulator with a conserved binding signal in eubacteria and archaea.