Stomatal opening is stimulated by light, including blue and red light. Blue light-induced stomatal opening is fairly well understood: it is mediated by blue-light photoreceptor phototropins (phot1 and phot2). Blue light activates the plasma membrane (PM) H+-ATPase via phosphorylation of its penultimate C-terminal residue. In addition, blue light induces starch degradation, resulting in the accumulation of sugars in guard cells. In Arabidopsis, all isogenes of PM H+-ATPase (AHA1–AHA11) are expressed in guard cells. Among them, AHA1 is the major isoform and is responsible for blue light-induced stomatal opening. Red light-induced stomatal opening, on the other hand, is thought to be dependent on photosynthesis in both guard cell chloroplasts and mesophyll cells; however, the mechanism by which red light induces stomatal opening and whether PM H+-ATPases are involved in this process are controversial. Ando and Kinoshita (10.1104/pp.18.00544)have established an immunohistochemical technique to detect the phosphorylation level of PM H+-ATPase in guard cells using whole leaves of Arabidopsis. Unexpectedly, they found that red light induces phosphorylation of the penultimate C-terminal residue of PM H+-ATPase in whole leaves and this process is inhibited by the plant hormone abscisic acid. Red light-dependent stomatal opening was delayed in whole leaves of aha1-9, a knockout mutant of one of the major isoforms of PM H+-ATPase in guard cells. Furthermore, the photosynthetic electron transport inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), inhibited red light-induced PM H+-ATPase phosphorylation, as well as red light-induced stomatal opening in whole leaves. These results indicate that red light-induced PM H+-ATPase phosphorylation in guard cells promotes stomatal opening in whole leaves, providing insight into the photosynthetic regulation of stomatal opening.