Recent studies have suggested that Ca2+/calmodulin (CaM) or CaM-like proteins may be involved in blue light (BL)-dependent proton pumping in guard cells. As the increase in cytosolic concentration of Ca2+ is required for the activation of CaM and CaM-like proteins, the origin of the Ca2+ was investigated by measuring BL-dependent proton pumping with various treatments using guard cell protoplasts (GCPs) from Vicia faba. BL-dependent proton pumping was affected neither by Ca2+ channel blockers nor by changes of Ca2+ concentration in the medium used for the GCPs. Addition of Ca2+ ionophores and an agonist to GCPs did not induce proton pumping. However, BL-dependent proton pumping was inhibited by 10 mM caffeine, which releases Ca2+ from the intracellular stores, and by 10 μM 2,5-di-(tert-butyl)-1,4-benzohydroquinone (BHQ) and 10 μM cyclopiazonic acid (CPA), inhibitors of Ca2+-ATPase in the sarcoplasmic and endoplasmic reticulum (ER). By contrast, the inhibitions were not observed by 10 μM thapsigargin, an inhibitor of animal ER-type Ca2+-ATPase. The inhibitions by caffeine and BHQ were reversible. Light-dependent stomatal opening in the epidermis of Vicia was inhibited by caffeine, BHQ, and CPA. From these results, we conclude that the Ca2+ thought to be required for BL-dependent proton pumping may originate from intracellular Ca2+ stores, most likely from ER in guard cells, and that this origin of Ca2+ may generate a stimulus-specific Ca2+ signal for stomatal opening.