Neomycins belong to a family of clinically important 2-deoxystreptamine (2DOS) containing aminoglycoside antibiotics including kanamycin, gentamicin, tobtamycin, and butirosin. In 2005, biosynthetic genes for neomycin were identified from the producer strain Streptomyces fradiae and the functions of those gene products have been extensively investigated with recombinant proteins. Among seven conserved enzymes encoded in the neomycin, butirosin, kanamycin, gentamicin, and tobramycin biosynthetic gene clusters, NeoC, NeoS and NeoE were already reorted to be responsible for the construction of 2DOS from D-glucose 6-phosphate (G6P). In the present study, a putative glycosyltransferase NeoM was characterized as UDP-GlcNAc:2DOS GlcNAc transferase. Comparative analysis of aminoglycoside biosynthetic genes revealed that four conserved ribostamycin-related enzymes couls be responsible for the ribosylation of neamine. As a result of enzymatic analysis with recombinant proteins derived from the butirosin producer Bacillus circulans, we verified that BtrL (NeoL homolog) catalyzes phosphoribosylation of ribostamycin, and BtrP (NeoP homolog) catalyzes following dephospholylation giving ribostamycin. Two conserved neomycin-related enzymes seemed to be involved in the last glycosylation in neomycin biosynthesis. One of the enzymes NeoF was then found to catalyze the GlcNAc transfer reation onto ribostamysin. The NeoF reaction product was further deacetylated by repetitive functional N-Ac-paromamine deacetylase NeoD, followed by dehydrogenation (NeoQ) and transamination (NeoB) leading to neomycin C. In addtion to previous results from our and British groups, all biosynthetic enzymes to synthesize neomycin C from G6P have been finally characterized.