The aim of this study was to examine the frequency and predictors of colonization of the respiratory tract by metallo-beta-lactamase (MBL)-producing Gram-negative bacteria in patients admitted to a newly established intensive care unit (ICU) of a tertiary care hospital. Specimens of tracheobronchial aspirates for microbiological studies were obtained every day for the first 3 days of the ICU stay and subsequently every third day for the rest of the ICU stay. PCR analysis and nucleotide sequencing were performed to identify bacteria that had MBL genes. Thirty-five patients (20 male, 15 female) were hospitalized during the initial 3 month period of functioning of the ICU. Colonization of the lower respiratory tract by Gram-negative bacteria was found in 29 of 35 patients (83 %) during the first 6-20 days (median 13 days) following admission to the ICU (13 patients with Acinetobacter baumannii, ten with Pseudomonas aeruginosa, three with Enterobacter aerogenes, two with Klebsiella pneumoniae and one with Stenotrophomonas maltophilia). Six of 29 patients (21 %) colonized with Gram-negative bacteria had bla(VIM-2)-positive P. aeruginosa isolates; one of these patients developed clinical infection due to this micro-organism. Previous use of carbapenems (P=0.01) or other beta-lactams (P=0.03), as well as a stay in the ICU of >20 days (P<0.001), were associated with colonization with bla(VIM-2)-producing P. aeruginosa. In conclusion, colonization by Gram-negative bacteria of the respiratory tract of patients in this newly established ICU was common (83 %). Use of beta-lactams, including carbapenems, was associated with subsequent colonization of the respiratory tract with MBL-positive P. aeruginosa.