Carbapenem resistance among Pseudomonas aeruginosa and Acinetobacter spp. is becoming a critical therapeutic problem worldwide. The SENTRY Antimicrobial Surveillance Program monitors pathogen frequency and antimicrobial resistance patterns of nosocomial and community-acquired infections through sentinel hospitals on five continents. Pseudomonas spp. and Acinetobacter spp. strains resistant to imipenem (MIC, >/=16 mg/l), meropenem (MIC, >/=16 mg/l), and ceftazidime (MIC, >/=32 mg/l) collected from January 2001 to December 2003 were routinely screened for antimicrobial resistance genes. Resistant isolates were initially tested for metallo-beta-lactamase (MbetaL) production by phenotypic tests (disk approximation or MbetaL Etest strip) and then characterization of the MbetaL (hydrolysis assays, PCR for bla(IMP), bla(VIM), bla(SPM), gene sequencing). Eighty-nine isolates (33 Acinetobacter spp., 54 Pseudomonas aeruginosa, and 2 P. fluorescens) had positive phenotypic screening tests. Among those, 34 isolates producing MbetaL were identified, including 7 Acinetobacter spp., 25 P. aeruginosa and 2 P. fluorescens. The MbetaLs identified were IMP-1, VIM-2 and two newly described enzymes: SPM-1 and IMP-16. The greatest concentration of MbetaL strains was in Brazil, where imipenem-resistant P. aeruginosa increased significantly in the time period evaluated by the SENTRY Program. MbetaL-producing P. aeruginosa was detected in São Paulo (SPM-1) and Brasilia (SPM-1 and IMP-16), Brazil and Caracas, Venezuela (VIM-2); while MbetaL-producing Acinetobacter spp. isolates were detected in São Paulo, Brazil (IMP-1). P. fluorescens isolates producing IMP-1 and VIM-2 were detected in São Paulo, Brazil and Santiago, Chile, respectively. The emergence and dissemination of mobile MbetaL-producing isolates represent an alarming factor for increasing resistance to carbapenems in several medical centres evaluated by the SENTRY Program in Latin America.