An in vitro protein-synthesizing system can synthesize two ribonucleic acid (RNA) polymerase subunits of Escherichia coli, beta and beta', when a transducing phage deoxyribonucleic acid (DNA) template containing the rpoB region of the bacterial chromosome is added. Recombinant rpoB transducing phages were isolated that carry "nonsense" mutations of the class rpo-rifampin zero amber (formally referred to as rifoam). DNA was extracted from two of these phages. These DNAs are unable to direct the synthesis of beta subunits, whereas beta' synthesis is unaffected. Both mutations can be efficiently suppressed in vitro by the addition of suppressor transfer RNA. One of the mutations (rpoB115) produces a detectable nonsense (or restart) fragment of the beta protein in the absence of suppression. It is concluded that rpoB115 is an amber mutation within the structural gene for the beta subunit of RNA polymerase.