The Escherichia coli B gene (nfnB) encoding nitroreductase has been cloned in Escherichia coli K-12 and its nucleotide sequence determined. The translated amino acid sequence was found to share substantial identity (88.5%) with the equivalent proteins of Enterobacter cloacae and Salmonella typhimurium. When the structural gene was placed under the transcriptional control of either the trp or lac promoter, recombinant nitroreductase was accumulated to 33% and 25% of the cell's soluble protein, respectively. Substitution of the nfrB ribosome binding site with that of the E. coli lacZ gene reduced production levels of nitroreductase. The sequenced region also contained two incomplete open reading frames of unknown function.