Enterobacter species were studied longitudinally in a children's hospital. In total, 287 Enterobacter isolates were obtained from 171 children in 15 different wards (from March 1995 through April 1997). Strains were typed by random amplified polymorphic DNA and pulsed-field gel electrophoresis, which were concordant in outcome. In total, 97 DNA types and 199 colonization events were identified. A predominant clone was isolated 111 times from 62 children; another clone was isolated 19 times from 10 patients. These clones caused 36% of all colonizations. In 34% of the children, Enterobacter clones were found in 2-4 patients. The remaining colonizations were due to unique Enterobacter isolates. A large proportion of the Enterobacter strains was acquired through cross-transmission. This finding contrasts with the prevailing opinion that resistant Enterobacter strains are selected primarily from the patient's own gut flora.