Pseudomonas aeruginosa GW-1 was isolated in 2000 in South Africa from blood cultures of a 38-year-old female who developed nosocomial pneumonia. This isolate harbored a self-transferable ca. 100-kb plasmid that conferred an expanded-spectrum cephalosporin resistance profile associated with an intermediate susceptibility to imipenem. A beta-lactamase gene, bla(GES-2), was cloned from whole-cell DNA of P. aeruginosa GW-1 and expressed in Escherichia coli. GES-2, with a pI value of 5.8, hydrolyzed expanded-spectrum cephalosporins, and its substrate profile was extended to include imipenem compared to that of GES-1, identified previously in Klebsiella pneumoniae. GES-2 activity was less inhibited by clavulanic acid, tazobactam and imipenem than GES-1. The GES-2 amino acid sequence differs from that of GES-1 by a glycine-to-asparagine substitution in position 170 located in the omega loop of Ambler class A enzymes. This amino acid change may explain the extension of the substrate profile of the plasmid-encoded beta-lactamase GES-2.