Among gram-negative bacilli isolated from burn wound cultures, some strains of Enterobacteriaceae were resistant to tobramycin (minimal inhibitory concentration [MIC]>/= 20 mug/ml) but susceptible to gentamicin (MIC </= 5 mug/ml). One Klebsiella pneumoniae and two Enterobacter cloacae strains were selected for studies on their mechanisms of resistance to aminoglycoside antibiotics. Resistance to high concentrations of tobramycin (MICs of 25 to 50 mug/ml) was conjugally transferred to a susceptible Escherichia coli strain at rates of 1.2 x 10(-4) to 2.8 to 10(-4) per donor cell, suggesting that resistance is controlled by R factors. Resistances to tobramycin, kanamycin, and neomycin were cotransferred. Enzymatic activities were present that acetylated tobramycin, gentamicin, and kanamycin in osmotic lysates from the donor and transcipient strains. Enzymatic adenylylation of these aminoglycosides was not observed. The aminoglycoside-acetylating activities from K. pneumoniae and E. cloacae resembled kanamycin acetyltransferase (KAT) in their specificity for aminoglycoside substrates. Not all isolates of bacteria that produce KAT are resistant to tobramycin, but the factors that determine susceptibility or resistance to tobramycin in KAT-producing bacteria have not yet been established.