The prevalence of imipenem resistance among Pseudomonas aeruginosa isolates at a 195-bed tertiary care medical center in Cali, Colombia, rose from 2% in 1996 to 28% in 1997 and to over 40% in 2003. Many isolates showed high-level multiresistance, and phenotypic characterization suggested the spread of a predominant strain with minor variants. Sixty-six resistant isolates collected between February 1999 and July 2003 from hospitalized patients (n = 54) and environmental samples (n = 12) were subjected to a fuller analysis. Genetic fingerprints were compared by pulsed-field gel electrophoresis (PFGE) of SpeI-digested genomic DNA, and blaIMP and blaVIM genes were sought by PCR. PFGE and serotyping indicated that 52 of the 66 isolates belonged to a single strain, with 82% similarity; the PFGE pattern for this organism was designated pattern A. Two further pairs of isolates represented single strains; the remaining nine isolates were unique, and in the case of one isolate, no satisfactory PFGE profile could be obtained. The pattern A isolates were mostly of serotype O12 and were highly resistant to imipenem (MICs, 32 to >256 µg/ml), with this resistance decreased eightfold or more in the presence of EDTA. They yielded amplicons with blaVIM-specific primers, and sequencing of DNA from a representative isolate revealed blaVIM-8, a novel allele with three polymorphisms compared with the sequence of blaVIM-2. Two of these nucleotide changes were silent, but the third determined a Thr139Ala substitution. Only 4 of 13 resistant isolates (2 clinical isolates and 2 environmental isolates) assigned to other PFGE types carried blaVIM alleles, whereas the others were less multiresistant and mostly had lower levels of imipenem resistance (MICs, =32 µg/ml) which was not significantly reduced by EDTA. No blaIMP alleles were detected. During 2003, when the environmental study was undertaken, serotype O12 isolates with blaVIM were recovered from sinks and stethoscopes in the most-affected units, although not from the hands of staff; the problem declined once these reservoirs were disinfected and hygienic precautions were reinforced.