The prevalence of metallo-beta-lactamase (MBL) production among Pseudomonas aeruginosa nosocomial isolates from a Brazilian teaching hospital was determined. A total of 512 P. aeruginosa isolates were recovered from 245 patients during a 10-month period. Ninety-four (38.4%, 95% CI 32.2-44.8%) isolates were MBL producers. Most resistance to beta-lactams was mediated by MBL. Forty-one (16.7%) were resistant to all drugs except polymyxin B and 33 (80.5%) of these were MBL producers. Clonal dissemination, documented by DNA macrorestriction, played a major role for the spread of MBL isolates. The blaSPM-1 gene was demonstrated by PCR in 14 randomly selected MBL isolates. The extremely high prevalence of MBL production found challenges the choice of therapeutics for P. aeruginosa, and measures to control horizontal dissemination of MBL producers are urgently required.