A carbapenem-resistant Acinetobacter baumannii strain was isolated in Toulouse, France, in 2003. Cloning and expression in Escherichia coli identified the carbapenem-hydrolyzing beta-lactamase OXA-58, which is weakly related (less than 50% amino acid identity) to other oxacillinases. It hydrolyzed penicillins, oxacillin, and imipenem but not expanded-spectrum cephalosporins. The bla(OXA-58) gene was located on a ca. 30-kb non-self-transferable plasmid. After electrotransformation in the A. baumannii CIP7010(T) reference strain, it conferred reduced susceptibility to carbapenems. The bla(OXA-58) gene was bracketed by two novel ISAba3-like insertion elements. This study describes a newly characterized beta-lactamase that may contribute to carbapenem resistance in A. baumannii.