The DNA of the attenuator region of the histidine operon of Escherichia coli has been transcribed in a purified in vitro system and found to synthesize two major RNA transcripts. The first one, 180 nucleotides long, has been identified as the histidine-specific leader RNA. It contains the coding sequence for the leader peptide [Di Nocera, P. P., Blasi, F., Di Lauro, R., Frunzio, R. & Bruni, C. B. (1978) Proc. Natl. Acad. Sci. USA 75, 4276-4280] and is terminated at the attenuator site. Termination of transcription at this site is extremely efficient in the in vitro system. The leader RNA also has been detected in vivo in a minicell producer strain transformed with plasmids harboring the regulatory region of the histidine operon of E. coli. A second RNA molecule is synthesized in the in vitro system. It has a divergent direction of transcription with respect to the histidine leader RNA, but its role, if any, in the regulation of the histidine operon remains to be ascertained. The existence of the histidine leader RNA lends support to the regulatory mechanism which postulates that regulation of the histidine operon is dependent on the alternative secondary structures that the leader RNA may assume, depending on whether or not the histidine-rich leader peptide is translated.