The reconstitution of high O 2 evolution in CaCl 2 -treated PS II particles was achieved by the simultaneous addition of the 17-, 23- and 34-kDa proteins and total thylakoid lipids in the presence of 25% glycerol and 15 mM sodium cholate. The activity of the reconstituted membranes recovered to 85% of that of the non-depleted original PS II particles at the optimal condition. By means of this reconstitution method, evidence for the cooperation of the three proteins in the recovery of O 2 evolution in the CaCl 2 -treated PS II particles was found by changing the concentration of NaCI in the assay medium, and the relationship between the amount of manganese retained in the water-splitting complex and the O 2 evolving activity was examined by using the partially solubilized PS II particles with n-octyl-β-D-glucoside.