The regulation of pyrG expression in a group of low GC Gram-positive bacteria was previously shown to be mediated by a novel form of transcription attenuation in which low levels of intracellular CTP induce reiterative addition of G residues at position +4 in the 5' end of the pyrG mRNA, which is encoded as pppGGGC. . . . The poly(G) sequences formed under these conditions act to prevent attenuation by base pairing with the C- and U-rich 5' strand of a downstream terminator stem-loop located in the pyrG leader. In this work we document the reconstitution of this regulatory system in vitro using only the native pyrG DNA template, RNA polymerase and appropriate concentrations of ribonucleotides. CTP-regulated reiterative transcription producing 5'-poly(G) tracts and regulation of transcription termination at the pyrG attenuator by CTP were demonstrated. Mutations in the native pyrG template that altered reiterative transcription and attenuation in vivo resulted in alternations in expression in the in vitro transcription system that were predicted by the mechanism described above. These findings provide strong experimental support for the proposed reiterative transcription/antitermination mechanism and confirm that no trans-acting regulatory protein is required for pyrG regulation.