Despite the most of the 2-deoxystreptamine (2-DOS) containing aminoglycosides are produced from Streptomyces, very little information exists about their biosynthetic genes; cloning and characterization. 2-deoxy-scyllo-inosose synthase (2-DOIS) was isolated from S. kanamyceticus and expressed in heterologous host. We have isolated 2-DOS biosynthetic genes from several aminoglycoside producers Streptomyces and Micromonospora species; 2-deoxy-scyllo-inosose synthase, 2-deoxy-scyllo-inosose aminotransferase and aminoglycoside resistance ribosomal methylases, by PCR experiment using the primers designed from the conserved region of respective genes. The sequencing of PCR amplified DNA fragments reveled their good homology with btrC and btrB of Bacillus circulans, the sole 2-DOIS gene exists in the database until now, and with other aminoglycodide resistance ribosomal methylases. As a goal for isolation of biosynthetic gene cluster of tobramycin, a cosmid, pST51 was isolated and sequencing of this cosmid is under progress. The PCR primers are specific and reliable to isolate the biosynthetic genes of 2-DOS containing aminoglycosides or for screening the aminoglycoside producers. The use of 2-DOIS as probe can save both time and cost for cloning aminoglycoside biosynthetic genes.