Proton matrix ENDOR spectra were measured for Ca2+-depleted and Sr2+-substituted photosystem II (PSII) membrane samples from spinach and core complexes from Theymosynechococcus vulcanus in the S-2 state. The ENDOR spectra obtained were similar for untreated PSII from T. vulcanus and spinach, as well as for Ca2+-containing and Sr2+-substituted PSII, indicating that the proton arrangements around the manganese cluster in cyanobacterial and higher plant PSII and Ca2+-containing and Sr2+-substituted are similar in the S-2 state, in agreement with the similarity of the crystal structure of both Ca2+-containing and Sr2+-substituted PSII in the Si state. Nevertheless, slightly different hyperfine separations were found between Ca2+-containing and Sr2+-substituted PSII because of modifications of the water protons ligating to the Sr2+ ion. Importantly, Ca2+ depletion caused the loss of ENDOR signals with a 1.36-MHz separation because of the loss of the water proton W4 connecting Ca2+ and Y, directly. With respect to the crystal structure and the functions of Ca2+ in oxygen evolution, it was concluded that the roles of Ca2+ and Sr2+ involve the maintenance of the hydrogen bond network near the Ca2+ site and electron transfer pathway to the manganese cluster.