An oxygen-evolving, Photosystem II particle was isolated from the thermophilic, blue-green alga, Phormidium laminosum, according to the procedure of Stewart and Bendall (Stewart, A.C. and Bendall, D. (1979) FEBS Lett. 107, 308–312). Our particle has an oxygen-evolution activity of 1500–1600 μmol O 2/mg chlorophyll per h. The oxygen-evolution activity has a pH optimum at 5–6, and is abolished at pH 9. Maximum oxygen evolution occurs at approx. 47°C in whole cells, but at 29°C in the particles. The activity decreases to 50% when the cells are heated for 30 min at 55°C; with the particles, 50% inactivation occurred at 47°C for the same heating time of 30 min. Flash excitation of the particle at 100 K produced absorbance changes whose difference spectrum in the ultraviolet-to-near infrared region shows photochemical charge separation and recombination of P-680 + and Q − in the dark with t 1 2 of 1.75 ms. An EPR spectrum for the P-680 + free radical, with g 2.0027 and ΔH pp = 8 G, was constructed from flash-induced EPR changes under conditions identical to those used for obtaining P-680 absorbance changes. The actinic light-induced variable fluorescence yield is 5-fold that induced by the weak probing beam alone. Addition of dithionite to the particle brings the fluorescence to the same maximum level. Under the reducing condition, strong actinic light caused the fluorescence to decrease. This observation is consistent with the notion that variable fluorescence yield in Photosystem II originates, as in green-plant chloroplasts, from recombination luminescence, the attenuation of which corresponds to photoaccumulation of reduced pheophytin under these conditions. Broad segments (300 nm) of the difference spectrum for pheophytin photoreduction were recorded by an intensified photodiode array in conjunction with a phosphoroscopic photometer. Kinetic spectrophotometric assays together with chemical analysis showed a rather clean and simple stoichiometry in these particles, namely, 1 P-680:1 Ph:1 Q:4 Mn:44 Chl. Initial investigation failed to reveal the doublet EPR spectrum previously observed for Ph −·Q − Fe in spinach subchloroplast particles (Klimov, V.V., Dolan, E. and Ke, B. (1980). FEBS Lett. 118, 97–100). A hyperfine EPR spectrum consisting of 16–20 lines and presumably associated with the manganese clusters in the oxygen-evolving protein has been confirmed in these particles. Tris washing but not washing with EDTA eliminates this signal. Active oxygen-evolving particles also yield the II vf signal with a t 1 2 of approx. 800 μs. Upon Tris washing, the II f signal appears which decays in 23.5 ms.