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Methods for the isolation of highly active oxygen-evolving photosystem (PS)II membranes from higher plants and the purification of the oxygen-evolving complex (OEC) subunits are described. Membrane samples used as the material for various in vitro studies of PSII are prepared by solubilization of thylakoid membranes with the non-ionic detergent Triton X-100. The OEC subunit proteins are dissociated from the PSII-enriched membranes by alkaline treatment or salt treatment, and then purified by ion-exchange chromatography using an automated high-performance liquid chromatography system.