A genomic library of S. ribosidificus was constructed for sorting out the biosynthetic genes for 4,5-disubstituted 2-deoxystreptamine (DOI) containing amino glycosides antibiotics ribostamycin. Screening over 10, 000 colonies of the library by the conserved region based probes revealed 6 positive cosmids. Sequencing about 32 kb regions reveled 27 open reading frames (ORFs) putatively involved in ribostamycin biosynthesis. The genetic organization of these gene clusters is in good agreement with the previously reported butirosin biosynthetic genes cluster. Out of 10 putative Rbm biosynthetic genes, the rbmA was expressed in S. lividans TK24 and was shown to encode 2-deoxy-scylloinosose (DOI) synthase. Furthermore, acetylations of various aminoglycosideaminocyclitol (AmAcs) with RbmI concluded the rbml as an aminoglycoside 3-N-acetyltrasferase.