Pioneering work leading to the elucidation of the genetic code and the characterization of the protein synthesis machinery relied heavily on in vitro transcription and translation systems. Such systems allowed the use of defined templates to direct the synthesis of proteins, which could be directly characterized. It is these characteristics of the E. coli -coupled transcription/translation system (S-30) that are still useful for a variety of applications. The in vitro system, first described by Zubay (1 ), is the basis for the protocol described here.