Acinetobacter calcoaceticus subsp. anitratus (Herellea vaginicola) is an important cause of nosocomial infection in our hospital where A. calcoaceticus subsp. anitratus is the most frequently isolated gram-negative species resistant to one or more of the aminoglycoside antibiotics. Of 167 strains tested for susceptibility to aminoglycosides, only 6 strains were found that were resistant to >/=128 mug of kanamycin per ml; all others were susceptible to </=32 mug/ml. Five of these six strains were found to produce aminoglycoside-modifying enzymes. Two strains produced a phosphotransferase which mediates resistance to kanamycin and neomycin; three strains produced an acetyltransferase which mediates resistance to kanamycin, tobramycin, and amikacin (minimal inhibitory concentration >/= 128 mug/ml for each drug). No strain with lower level resistance to kanamycin, tobramycin, or amikacin had enzyme activity. Fourteen strains resistant to gentamicin failed to show significant enzymatic modification of that antibiotic. Although agarose gel electrophoresis of deoxyribonucleic acid preparations from the enzyme-producing strains showed plasmid bands in all, no transfer of aminoglycoside resistance could be achieved, nor was it cured by exposure to novobiocin, ethidium bromide, acridine orange, heat, or prolonged storage. Resistance to mercuric chloride, present in 2 of 60 strains, was lost by 1 strain after exposure to novobiocin, and the loss of resistance was associated with an apparent deletion of plasmid deoxyribonucleic acid.