Biotin synthase catalyses the final step in the biotin biosynthetic pathway and is encoded by the bioB gene in Escherichia coli. To investigate the conversion of dethiobiotin to biotin in the plant kingdom, the cDNA encoding the bioB gene product equivalent from Arabidopsis thaliana was used to construct an E. coli overexpression strain. The purified A. thaliana bioB gene product is a homodimer (100 kDa) with a reddish color and has an absorbance spectrum characteristic of protein with [2Fe-2S] clusters. Its intracellular compartmentation in pea leaves discloses a unique polypeptide of 39 kDa within the matrix of mitochondria.