Abstract Incubation of PS II membranes at 45–50°C for several min resulted in strong inactivation of oxygen evolution, concomitant with release of Mn and the extrinsic proteins of 33, 23 and 17 kDa. No correlation was found between loss of the activity and release of the 33 kDa protein or Mn. However, involvement of the protein release in the mechanism of heat-inactivation was suggested by stabilization of the activity against heat-treatment by immobilization of the 33 kDa protein with a water-soluble carbodiimide. Furthermore, a linear correlation was found between extents of heat-inactivation and amounts of the 33 kDa protein released in the presence of 50 mM CaCl 2, which greatly accelerated inactivation of oxygen evolution, release of the 33 kDa protein and aggregation of PS II membranes at high temperatures. Evidence was obtained indicating that the 33 kDa protein released at high temperatures rebinds to its functional site when temperature is lowered but CaCl 2 suppresses rebinding of the protein by promoting intensive aggregation of the membranes. Thus, the activity survived in the presence of CaCl 2 is proportional to the amounts of the protein remained attached to the membranes during heat-treatment. By contrast, release of Mn was not affected by addition of CaCl 2 so that enhanced inactivation of oxygen evolution was not accompanied by corresponding increase in the amount of Mn released. It is concluded, therefore, that the primary cause of heat-inactivation of oxygen evolution is dissociation of the 33 kDa protein but not that of Mn.