Forty-three intubated and mechanically ventilated patients in five intensive care units (ICUs) of one hospital developed respiratory colonization or infection with Acinetobacter calcoaceticus subspecies anitratus over a 16-month interval. Neither the frequency nor rate of A anitratus isolation exceeded the hospital endemic norms. Single isolates from 34 of the patients were subtyped by plasmid DNA analysis, two biotyping systems and antimicrobial susceptibility to 24 drugs. Plasmid DNA fingerprints were distinct in 18 isolates (they differed from each other and all others), similar in two and identical or similar in ten. The latter group of isolates were recovered from patients in four ICUs. Reproducibility of biotyping was poor. Neither biotyping nor antimicrobial susceptibility were successful in identifying sameness among the group isolates nor differences among other isolates. We conclude that plasmid DNA fingerprinting should be used to assess the possibility of multiple patient transmissions of the same A anitratus strain in the absence of an obvious outbreak.