A novel gene, bla(KHM-1), encoding a metallo-beta-lactamase, KHM-1, was cloned from a clinical isolate of Citrobacter freundii resistant to most beta-lactam antibiotics. Escherichia coli expressing bla(KHM-1) was resistant to all broad-spectrum beta-lactams except for monobactams and showed reduced susceptibility to carbapenems. Recombinant KHM-1 exhibited EDTA-inhibitable hydrolytic activity against most beta-lactams, with an overall preference for cephalosporins.