The large subunit of ribulose 1,5-bisphosphate carboxylase (rbcL) and the beta subunit of chloroplast ATP synthase (atpB) are encoded by divergently transcribed genes on the plastid genome. We have identified DNA binding factors specific for sequences located in the intergenic region between these two genes. Soluble plastid extracts from pea or whole cell extracts from maize protected a maize chloroplast DNA probe containing the 160-base pair region between the 5' ends of rbcL and atpB genes from exonuclease III digestion between positions -16 and -101 relative to the rbcL gene transcription start site. Competition assay with partial sequences from this intergenic region demonstrated that specific sequence(s) are required for the protection. The borders of the binding domain are conserved among the homologous regions of maize, tobacco, spinach, and pea chloroplast genomes. Gel filtration chromatography revealed a molecular weight of about 115,000 for the active complex involved in DNA binding. Using the exonuclease III protection assay, we have also shown that purified Escherichia coli RNA polymerase protects from +25 to -20 of the rbcL gene and from +21 to -23 of the atpB gene relative to their respective transcription start sites. These regions are analogous to open complexes found when E. coli RNA polymerase interacts with the prokaryotic promoters and are consistent with the ability of E. coli RNA polymerase to initiate transcription correctly on linear templates containing these chloroplast promoters. Possible role(s) for the chloroplast DNA binding factor in chloroplast gene expression and its regulation are discussed.