Genomic libraries of S. ribosidificus and S. fradiae were constructed for sorting out the biosynthetic genes for 4,5 disubstitued-2-deoxystreptamine (DOI) containing amino glycosides antibiotics. Screening over 10,000 colonies of each library by the conserved region - based probes revealed 6 positive cosmids. Sequencing about 40 kb-region reveled 25 and 29 open reading frames (ORFs) putatively involved in ribostamycin and neomycin biosynthesis. The genetic organization of these gene clusters is in good agreement with the previously reported butirosin biosynthetic genes cluster. Furthermore acetylation by RbmJ of several amino glycosides was also conformed by in vitro enzyme assay of the purified protein.