Carbapenem resistance mediated by acquired carbapenemase genes has been increasingly reported, particularly for clinical isolates of Pseudomonas aeruginosa and Acinetobacter spp. Of 1,234 non-duplicate isolates of carbapenem-resistant Pseudomonas spp. and Acinetobacter spp. isolated at a tertiary-care hospital in Seoul, Korea, 211 (17%) were positive for metallo-beta-lactamase (MBL). Of these, 204 (96%) had either the bla(IMP-1) or bla(VIM-2) allele. In addition, seven Acinetobacter baumannii isolates were found to have a novel MBL gene, which was designated bla(SIM-1). The SIM-1 protein has a pI of 7.2, is a new member of subclass B1, and exhibits 64 to 69% identity with the IMP-type MBLs, which are its closest relatives. All SIM-1-producing isolates exhibited relatively low imipenem and meropenem MICs (8 to 16 microg/ml) and had a multidrug resistance phenotype. Expression of the cloned bla(SIM-1) gene in Escherichia coli revealed that the encoded enzyme is capable of hydrolyzing a broad array of beta-lactams, including penicillins, narrow- to expanded-spectrum cephalosporins, and carbapenems. The bla(SIM-1) gene was carried on a gene cassette inserted into a class 1 integron, which included three additional cassettes (arr-3, catB3, and aadA1). The strains were isolated from sputum and urine specimens from patients with pneumonia and urinary tract infections, respectively. All patients had various underlying diseases. Pulsed-field gel electrophoresis of SmaI-digested genomic DNAs showed that the strains belonged to two different clonal lineages, indicating that horizontal transfer of this gene had occurred and suggesting the possibility of further spread of resistance in the future.