The Mr 10,000 phosphoprotein was purified from photosystem II particles by solubilization of the particles in 5% (w/v) dodecyl dimethylamine oxide, centrifugation in 10% (w/v) sucrose, and three chromatography steps. The purified phosphoprotein showed a unique NH2 terminus indicating a highly purified polypeptide. The amino acid sequence for the first nine residues is NH2-Ala-Thr-Gln-Thr-Val-Glu-Ser-Ser-Ser . . . COOH. The amino acid composition was determined and could also be used to help distinguish the polypeptide from other known thylakoid proteins. The sequence and composition data indicated that the Mr 10,000 phosphoprotein is neither the hydrophobic 8-kDa subunit of the energy coupling complex nor cytochrome b-559, but rather a unique, as yet unidentified, polypeptide associated with photosystem II.