Infections due to multidrug- and carbapenem-resistant isolates of Acinetobacter baumannii were frequent among patients treated in the Tzannion General Hospital in Piraeus, Greece, throughout the period 2005 to 2006. Six of these isolates, derived from blood cultures of patients in three different wards (four were from patients in the intensive care unit) and taken at least 1 month apart from each other, were studied. Species identification, initially performed with the API 20NE system (bioMerieux, Marcy l'Etoile, France), was confirmed by sequencing of 16S rRNA genes. Antimicrobial MICs were determined by agar dilution. Screening for metallo-β-lactamases (MBLs) was performed with imipenem-EDTA synergy tests: (i) the MBL Etest (AB Biodisk), (ii) the double-disc synergy test, and (iii) the combined-disc test. β-Lactamases were studied by isoelectric focusing of crude cell extracts derived by sonication. The carbapenemase activity of β-lactamase-containing extracts was assessed by spectrophotometry using imipenem (100 μM) as the reporter substrate. The blaOXA and MBL genes were identified by PCR and sequencing. The isolates were typed by pulsed-field gel electrophoresis (PFGE). The location of the VIM-1-encoding integrons was determined by an I-CeuI-based method. The isolates were highly resistant to piperacillin-tazobactam, cefotaxime, ceftazidime, cefepime, aztreonam, ampicillin-sulbactam, gentamicin, amikacin, tobramycin, and ciprofloxacin, with imipenem and meropenem MICs ranging from 16 to 32 μg/ml. Only one isolate was MBL positive by the double-disc synergy test, though it tested negative by the MBL Etest and the combined-disc test; the remaining five isolates were negative by all three tests. The isolates exhibited similar PFGE patterns (80% relatedness), suggesting a clonal relationship. They carried the carbapenemase genes blaOXA-51, blaOXA-58, and blaVIM-1, produced a β-lactamase with a pI equal to that of VIM-1 (5.1) inhibited in situ by EDTA (indicating MBL expression), and contained class 1 integrons: In-Ab10a (with aacA4 and blaVIM-1) and In-Ab10b (with blaVIM-1), both located chromosomally. The findings indicate a protracted hospital outbreak caused by genetically related A. baumannii isolates producing VIM-1. Conventional EDTA-based tests may not be suitable for detecting these isolates, and the failure of phenotypic tests to detect VIM-1 underscores the role of multiple mechanisms, such as the production of OXA enzymes, in determining carbapenem resistance in this species.