The newly introduced semisynthetic aminoglycoside antibiotics, i.e., 3',4'-dideoxykanamycin B (DKB), 6'-N-methyl DKB (6'-Me-DKB) and amikacin (AK) have been found to be effective against gram-negative pathogens including Pseudomonas aeruginosa, which are resistant to the known aminoglycoside antibiotics. We have demonstrated in our stock cultures two types of P. aeruginosa strains resistant to DKB, i.e., (DKB(r).AK(r).6'-Me-DKB(s)) and (DKB(r).AK(s).6'-Me-DKB(r)) (where r = resistant; s = sensitive). Both groups of strains inactivate the drugs by acetylation. The acetylating enzyme was extracted from GN4925(DKB(r).AK(s).6'-Me-DKB(r)) and purified by affinity chromatography. Enzymatic studies of the inactivation reaction and chemical studies of the inactivated products indicated that DKB and 6'-Me-DKB were inactivated by acetylation of the 6'-amino group of the drugs. This enzyme acetylates kanamycin A (KM-A), KM-B, DKB, 6'-Me-DKB, 6'-N-methyl kanamycin B, but not KM-C, AK, and gentamicin C(1). The enzyme is named aminoglycoside 6'-N-acetyltransferase 3. Genetic studies of two strains resistant to DKB and 6'-Me-DKB disclosed that the enzyme catalyzing inactivation of both DKB and 6'-Me-DKB was mediated by an R factor, i.e., R(ms167) and R(ms168), capable of conferring resistance to KM, DKB, and 6'-Me-DKB, in addition to resistance to gentamicin, streptomycin, and sulfanilamide, and resistance to tetracycline, chloramphenicol, streptomycin and sulfanilamide respectively.