Carbapenems such as imipenem are extended-spectrum beta-lactam antibiotics, which are not hydrolyzed by the beta-lactamases commonly found in Enterobacteriaceae. Here we report a gene encoding a carbapenemase, which was cloned from the chromosome of a clinical isolate of Enterobacter cloacae, strain NOR-1, into pACYC184 plasmid in Escherichia coli. Unlike all the sequenced carbapenemases, which are class B metallo-beta-lactamases, the mature protein (NmcA) is a class A serine beta-lactamase. NmcA shares the highest amino acid identity (50%) with the extended-spectrum class A beta-lactamase MEN-1 from E. coli. In the opposite orientation from the nmcA promoter, an overlapping and divergent promoter was detected, along with an open reading frame, which encoded a 33.5-kDa protein (NmcR). The NmcR amino acid sequence displays homology with LysR-type transcriptional regulatory proteins, including the conserved residues near its N terminus within a helix-turn-helix motif. Deletion of nmcR resulted in decreased carbapenem resistance and a loss of beta-lactamase inducibility, demonstrating a positive role of NmcR in NmcA expression.