The photosystem-II-associated 33-kDa extrinsic manganese-stabilizing protein is found in all oxygen-evolving organisms. In this paper, we show that this protein undergoes pH-induced conformational changes in the physiological pH range. At a neutral pH of 7.2, the hydrophobic amino acid residues that are most likely located inside the β barrel are "closed" and the protein binds neither Mn2+ nor Ca2+ ions. When the protein is transferred to a solution with a slightly acidic pH of 5.7, hydrophobic amino acid residues become exposed to the surrounding medium, enabling them to bind the fluorescent probe 8,1-ANS. At this pH-induced open state, Mn 2+ and Ca2+ bind to the manganese-stabilizing protein. The pH values used in this study, 7.2 and 5.7, are typical of the pH found in the thylakoid lumen in the dark and light, respectively. A model is presented in which the manganese-stabilizing protein undergoes a pH-dependent conformational change that in turn influences its capacity to bind calcium and manganese. In this model, the proton-dependent conformational changes of the tertiary structure of the manganese-stabilizing protein are of functional relevance for the regulation of substrate (water) delivery to and product (proton) release from the water-oxidizing complex by forming a proton-sensing proton-transport pathway.