Plasmid pRSP1, isolated from the Escherichia coli gene bank of Clarke and Carbon (1975), has been shown to complement the gdh-1 mutation that affects the synthesis of glutamate dehydrogenase (GDH) in the E. coli strain PA340. The GDH structural gene of E. coli is present on a 2.3-Md DNA fragment from the hybrid plasmid pRSP1 and certain of its derivatives. Polypeptides synthesized by minicells carrying some of these plasmids, enabled us to make a preliminary determination of the direction of transcription of the gdh gene on the cloned fragments.