Among the σ70 family bacterial σ factors, group 2 σ factors have similar promoter recognition specificity to group 1 (principal) σ factors and express and function under specific environmental and physiological conditions. In general, the cyanobacterial genome encodes more than four group 2 σ factors, and the unicellular Synechococcus elongatus PCC 7942 (Synechococcus) has five group 2 σ factors (RpoD2-6). In this study, we analyzed expression of group 2 σ factors of Synechococcus at both mRNA and protein levels, and we showed that the rpoD3 expression was activated only by high light (1,500 μmol photons m -2 s-1) among the various stress conditions examined. After high light shift, rpoD3 mRNA accumulated transiently within the first 5 min and diminished subsequently, whereas RpoD3 protein increased gradually during the first several hours. We also found that the rpoD3 deletion mutant rapidly lost viability under the same conditions. Analysis of the rpoD3 promoter structure revealed the presence of an HLR1 (high light-responsive element 1) sequence, which was suggested to be responsible for the high light-induced transcription under the control of the NblS (histidine kinase)-RpaB (response regulator) two-component system (Kappell, A. D., and van Waasbergen, L. G. (2007) Arch. Microbiol. 187, 337-342), at +6 to +23 with respect to the transcriptional start site. Here we demonstrated that recombinant RpaB protein specifically bound to HLR1 of the rpoD3 and hliA genes in vitro, and overexpression of a truncated RpaB variant harboring only the phosphoreceiver domain derepressed the transcription in vivo. Thus, we have concluded that phosphorylated RpaB are repressing the rpoD3 and hliA transcription under normal growth conditions, and the RpaB dephosphorylation induced by high light stress results in transcriptional derepression. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.