A 2.2-kb region of DNA containing the birA gene of Escherichia coli has been sequenced. The birA gene sequence predicts a 35.3-kDal [321 amino acids (aa)] bifunctional protein containing biotin-operon-repressor and biotin-holoenzyme-synthetase activities. Mutations, generated by random insertion of XhoI linkers, defined the extent of the gene. Mutations affecting one or more of five discernable properties of birA [Barker, D. and Campbell, A., J. Bacteriol., 143 (1980) 789-800] were mapped. Three mutations that result in temperature-sensitive (ts) growth, birA85, birA215, and birA879 mapped in the N-terminal two-thirds of the protein. The birA352 mutation, which partially complements birA215 and birA879, maps in the N-terminal third of the protein. Finally, birA361 maps closest to the amino terminus.