We have reported [Ifuku, O., Kishimoto, J., Haze, S., Yanagi, M. & Fukushima, S. (1992) Biosci. Biotechnol. Biochem. 56, 1780-1785] the enzymic conversion of dethiobiotin to biotin (catalyzed by the enzyme encoded by bioB) in cell-free extract of Escherichia coli which had been genetically engineered for high bioB expression. An unidentified protein(s) in addition to the bioB gene product is obligatory for this reaction. We have found that this protein was precipitated from the cell-free extract with poly(ethyleneimine), and we have purified it to homogeneity by a procedure which includes ammonium sulfate fractionation, DEAE-cellulose chromatography, gel filtration, and Mono Q chromatography. The apparent molecular mass of the purified protein was estimated to be about 21 kDa by SDS/PAGE. The N-terminal amino acid sequence of the purified protein was identical with that of E. coli flavodoxin. We conclude that flavodoxin is required for conversion of dethiobiotin to biotin in E. coli. Studies with purified flavodoxin and the fraction containing the bioB gene product suggested that protein(s) in addition to the bioB gene product and flavodoxin is also obligatory for the reaction.