The bioH gene, which is obligatory for biotin biosynthesis in Escherichia coli, was isolated as an 11-kb EcoRI fragment from E. coli by complementation for the growth of the bioH mutant. Deletion analysis of this fragment showed that the bioH gene was located within the 3.3-kb EcoRI-EcoRV fragment. Sequencing this fragment revealed the presence of two open reading frames, and a further complementation test showed that one of them codes for this gene. The molecular weight of the bioH gene product was about 25-kDa by the maxicell method, which was in good accordance with the molecular weight from the predicted nucleotide sequence. The promoter region of the bioH gene had no significant homology with that of the biotin operon and no operator sequence for the binding of the repressor protein (birA). LacZ-fusion analysis showed that the activity of this bioH promoter was not repressed by biotin, suggesting that the gene expression of the bioH is not regulated by the birA gene product, which acts as a repressor of the biotin operon. We attempted to increase the biotin productivity by the introducing the bioH gene into a high-copy plasmid in the presence of the biotin operon, but the productivity of the biotin and biotin intermediates (total biotin) significantly decreased. This indicated that the overexpression of the bioH gene reduces the level of a biotin precursor, although it is required for biotin biosynthesis.