Using the gene mapping membrane technique, we identified a gene (nemA) that encodes N-ethylmaleimide reductase in Escherichia coli. The open reading frame encodes a polypeptide of 365 amino acids with a molecular mass of 39,514 Da. The deduced amino acid sequence showed a high degree of homology (87% identical) with the pentaerythritol tetranitrate reductase of Enterobacter cloacae and the morphinone reductase of Pseudomonas putida (52% identical).