The organization of the 2-deoxystreptamine (DOS) biosynthetic gene cluster of Micromonospora echinospora has been determined. Sequencing of a 14.04 kb-region revealed twelve open reading frames (ORFs): four putative DOS biosynthetic genes (gtmA, B, C, and D), five amino sugars biosynthetic genes (gtmE, G, H, I, and gacB), two aminoglycoside resistance genes (gtmF and J) as well as a hypothetical ORF (gacA). One of the putative DOS biosynthetic genes, gtmA, was expressed in Escherichia coli, and the purified protein was shown to convert glucose-6-phosphate (G-6-P) to 2-deoxy-scyllo-inosose (DOI), a key step in DOS biosynthesis. In addition gtmJ was expressed in Streptomyces lividans and shown to confer gentamicin resistance. Thus gtmA and gtmJ are implicated in the biosynthesis of gentamicin and in resistance to it, respectively.