The 2-deoxystreptamine aglycon is a commonstructural feature found in aminocyclitol antibiotics including neomycin, kanamycin, tobramycin, gentamicin, sisomicin, butirosin and ribostamycin. Akey enzymeinvolved in the biosynthesis of the 2-deoxystreptamine moiety is 2-deoxy-1sic>'//< 9-inosose (DOI) synthase which catalyses the carbocycle formation from D-glucose-6-phosphate to 2-deoxy-scy//< 9-inosose. Therecent success of isolating the 2-deoxy-, s' cy//6>-inosose synthase from Bacillus circulans prompted us to clone the gene responsible for this important enzymeby the use of reverse genetics approach. With the aid of DNAprobes constructed on the basis of the amino-terminal sequence of the purified 42kDasubunit of the enzyme, the responsible gene btrC wassuccessfully cloned. Subsequently the btrC gene was heterologously expressed in Escherichia coll, and the 2-deoxy-scy//oinosose synthase activity of the recombinant polypeptide wasconfirmed by chemical analysis. The btrC gene encodes a protein composed of 368 amino acids with a molecular mass of 40.7 kDa. Ourprevious proposal for the similarity of 2-deoxy-scylloinosose synthase to dehydroquinate synthase has been confirmed onthe basis of their amino acid sequences. Significant differences in the sequences can also be observed however, particularly in the crucial substrate recognition regions. Comparison of the BtrC sequence with those ofbiosynthetic enzymesfor other related microbial products is also discussed.