Organisms encoding multidrug resistance genes are becoming increasingly prevalent. During a 2-month period (December, 2000, to January, 2001), 83 consecutive isolates of Enterobacter spp. were collected in our microbiology department. Antibiotic susceptibility was determined using the Vitek II automatic system. We selected strains with decreased susceptibility to extended-spectrum cephalosporins. The double-disk potentiation test was positive in 10 of these strains, indicating the presence of extended-spectrum beta-lactamases (ESBLs). Polymerase chain reaction (PCR), isoelectric focusing (IEF), and sequencing identified TEM 24 beta-lactamase in the 10 selected E. aerogenes. Random amplification of polymorphic DNA (RAPD-PCR) revealed the same clonal origin for all the strains tested and strongly suggest an outbreak of multidrug-resistant E. aerogenes. To follow up the trends in ESBLs-producing Enterobacter infections in the hospital over time, we repeated the study 1 year later (December, 2001, to February, 2002). Only three ESBLs-producing Enterobacter were found. All of them corresponded to the previously characterized clone.