We reported previously that chloroplast DNA binding factors bind to the rbcL promoter R2 (-33 to 229 from ATG) region in Zea mays. In this study, we investigated transcriptional activity in the high salt extracts of chloroplast, and identified rbcL R2 region-specific binding proteins in high-salt extracts. In-vitro transcription and dot blot assays revealed that the extract was transcriptionally active. An electrophoretic mobility shift assay (EMSA) showed that R2 region Binding Factors 1 and 2 (named RBF-1,2) bound specifically to the R2 region. RBF-1 and 2 were precipitated in 0 to 34% and 34 to 51% ammonium sulfate fraction, respectively; their binding activities were detected in 0.2 M and 0.4 M KCI fractions of DEAE cellulose chromatography, respectively. RBF-1 and RBF-2 were purified to about 140- and 66-fold from the chloroplast high-salt extract, respectively, and had specific binding activity to the R2 region. These factors may play an important role in regulating the transcription of rbcL.