The filamentous soil "bacterium, Streptomyces hygrosooplcus forma glebosus ATCC 1467 ( S. glebosus ), synthesizes the antibiotic bluensomycln, a monoguanidinated analogue of dihydrostreptomycin (DSM) Cell-free extracts of’ S, glebosus were found to synthesize mono- and diphosphorylated derivatives of dihydrostreptomycin. The diphosphorylated compound produced by S. glebosus differs from the diphosphorylated product (DSM-3 c,6-diP) produced by S. blklnlensls ATCC 1162, a streptomycin producer. The monophosphorylated dihydrostreptomycin is neither dihydrostreptomycin-6-p nor dihydrostreptomycin-3-P. It has the chromatographic mobilities and characteristics of dihydrostreptomycin-3-P. Dihydrostreptomycin-6-P, dihydrostreptomycln-3'6-P, dihydrostreptomycin-3'-Pt and the isolated monophosphorylated product could all be further phosphorylated to diphosphorylated dihydrostreptomycins by S. glebosus extracts. Streptomycin 6-kinase found in S. blklnlensls phosphorylated the monophosphorylated dihydrostreptomycin product from S. glebosus. The resulting dlpnosphorylated product had the chromatographic behavior of dihydrostreptomycln-3,6-dip No streptomycin-6-P phosphatase activity, which occurs in streptomycin producers, could he detected in S. glebosus. It is suggested that extracts of S. glebosus contain an ATPjdlhydrostreptomycin 3-phosphotransferase (3-kinase) (Reaction 1) and a somewhat less active ATPtdihydrostreptomycin 6-phosphotransferase (6-kinase) which phosphorylates the product of the 3-kinase (Reaction 2). MgATp + DSM 2ü-klnase ^ MgADp + DSM-3"-P (1) S. glebosus MgATP + DSM-3"-P -6~klnase ) MgADP + DSM-3,6-diP (2) S. glebosus If the structure of the product of Reaction 1 can be confirmed, for the first time a stable 3-kinase has been found in a Streptomyces. It is particularly curious, and might be significant, that Streptomyces which synthesize streptomycin contain a 3-kinase, whereas a strain which synthesizes a dihydrostreptomycin analogue contains a different kinase. Certain clinical isolates of E. coll and Pseudomonads containing R factors are resistant to streptomycin. The major streptomycin-inactivating reaction is the phosphorylation of streptomycin by a 3M-kinase. Therefore, S. glebosus might have a gene for a 3-kinase similar to that found in E. coll and Pseudomonads containing streptomycin-resistant plasmids. One evolutionary theory suggests that the two kinases are fragments of a dihydrostreptomycin biosynthetic pathway remaining in S. glebosus, the bluensomycin producer.