The NblS-RpaB signalling pathway, the most conserved two-component system in cyanobacteria, regulates photosynthesis and acclimatization to a variety of environmental conditions and is involved in negative regulation of high-light-induced genes. However, relevant regulatory details of the NblS-RpaB signalling pathway remain to be elucidated. We recently showed that the response regulator RpaB is regulated by specific (de)phosphorylation from the histidine kinase NblS and that RpaB and its phosphorylatable residue Asp56 are both required for viability of Synechococcus elongatus PCC 7942. We show here that the phosphorylated form of RpaB is present in cells growing under standard laboratory conditions and that high light stress affected the ratio of phosphorylated to non-phosphorylated RpaB. It also decreased the amount of rpaB transcripts without appreciably changing the total levels of RpaB. Quantitative Western blotting and confocal microscopy analyses were consistent with RpaB being a very abundant regulator, with nucleoid localization. A genetically engineered RpaB-GFP (green fluorescent protein) fusion protein rescued lethality of the rpaB null mutant, indicating that it was functional. This is, to our knowledge, the first study demonstrating in a cyanobacterium, and for a two-component response regulator, that the in vivo ratio of phosphorylated to non-phosphorylated protein changes in response to environmental conditions.