The major RNA polymerase from mustard chloroplasts is a multi-subunit enzyme consisting of core components and associated factors. Among the latter is a heterotrimeric factor named PTK (plastid transcription kinase) because of its serine/threonine-type protein kinase activity. PTK activity itself depends on its phosphorylation state. In addition, we show that it responds to glutathione but not to other redox-reactive reagents that were tested, and both glutathione and phosphorylation act antagonistically. Using a homologous in vitro system, we find that PTK selectively phosphorylates subunit(s) of plastid RNA polymerase and is involved in determining the level of faithful transcription from the chloroplast psbA promoter. Together, these results establish a role for phosphorylation and redox state in the regulation of plastid gene expression.