A chloroplast protein fraction with sigma-like activity [Bülow, S. & Link, G. (1988) Plant Mol. Biol. 10, 349-357], was further purified and characterized. Chromatography on heparin-Sepharose, DEAE-Sepharose and Sephacryl S-300 led to the separation of three sigma-like factors (SLF) polypeptides with Mr 67,000 (SLF67), 52,000 (SLF52) and 29,000 (SLF29). None of these polypeptides bind to DNA itself, but each one confers enhanced binding and transcriptional activity when added to Escherichia coli RNA-polymerase core enzyme and DNA fragments carrying a chloroplast promoter. SLF67, SLF52, and SLF29 differ in their ionic-strength requirements for activity. They each mediate the binding to promoters of the chloroplast genes psbA, trnQ, and rps16, with different efficiencies. It is suggested that chloroplast transcription in vivo might be controlled at least in part by these functionally distinct factors.