The bifunctional enzyme imidazoleglycerolphosphate dehydratase and histidinolphosphate phosphatase is encoded by the hisB gene. The fourth gene of the histidine operon, hisB, was cloned and mapped on a 2,300 base pair DNA fragment. In the present study we report the complete nucleotide sequence of the hisB gene of Escherichia coli. The gene is 1,068 nucleotides long and codes for a protein of 355 amino acids with an apparent molecular weight of 39,998 daltons. The protein product(s) of the hisB region of both Salmonella typhimurium and E. coli were identified by subcloning and expression in an in vitro translation system. In both organisms the hisB gene directed the synthesis of a single protein with an apparent molecular weight of 40,500 daltons, consistent with the data derived from the nucleotide sequence analysis.