Many papers have been presented on the enzymatic inactivation of the aminoglycosidic antibiotics by cell-free extracts obtained from drug-resistant strains of bacteria isolated from clinical specimens. Kanamycin is inactivated by two mechanisms responsible for the formation of 6-N-acetyl-kanamycin and 3'-phosphorylkanamycin. Similarly, streptomycin is inactivated by the formation of adenylylstreptomycin and phosphorylstreptomycin. These facts indicate that three mechanisms, i.e., phosphorylation, acetylation and adenylylation, involve in the inactivation of aminoglycoside antibiotics. It was reported previously that lividomycin, a new aminoglycosidic pentasaccharide antibiotic containing 2-deoxystreptamine, lacks the hydroxyl group at the C-3 position of the D-aminoglucose moiety and is not inactivated by a kanamycin-phosphorylating enzyme. This paper deals with the studies of anti-bacterial activity of lividomycin (LV) toward many strains of bacteria isolated from clinical specimens. The results are summarized as follows : 1) There are two types of LV-resistant strains. One group is capable of inactivating LV by phosphorylation and another group can not inactivate the drug.2) The inactivated product is a monophosphorylated LV.3) Chemical studies disclosed that the hydroxyl group at the 5″-position of the ribose moiety is phosphorylated.