Oxygen evolution in Photosystem 11 (PS 11) membranes which had been inactivated by treatment with a high concentration of NaCl in the light was restored by addition of a wide concentration of Ca 2+ and kinetic analysis showed the occurrence of two binding sites with different affinity for Ca 2+; a low- ( K m = 1–2 mM) and a high-affinity ( K m = 50–100 μM) site. Additionally, removal of contaminating Ca '+ by treatment with Chelex 100 revealed a very-high-affinity binding site for Ca`+ (Km = 4–10 μM). A substantial portion of the activity restored by Ca 2+ survived treatment with a chelating reagent, ethylene glycol bis(β-aminoethyl ether)- N, N′-tetraacetate, which removed Cat+ bound to all the three affinity sites. Thus, there are three reversible binding sites and one irreversible binding site for Ca 2+ in NaCl-washed membranes. The binding affinities of the reversible binding sites for Ca 2+ were estimated after correcting for contribution from the irreversible binding site. When PS II membranes had been treated with increasing concentrations of NaCl, the irreversible binding site was partly converted into the reversible binding site and a low-affinity interaction of Ca 2+ increased at the cost of high-affinity one. Addition of a low concentration of Triton X-100 to the washing medium also increased the high- and low-affinity sites and decreased the very-high-affinity site. Conversely, prolonged incubation of the salt-washed membranes with Ca 2 resulted in conversion of the high- and low-affinity sites to the very-high-affinity site. Thus, the four binding sites are at least partly interconvertible. The results show that the heterogeneity of Ca 2+-binding in terms of both binding affinity and reversibility derives from differently modified states of a single binding site.