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The cytochrome b 559 content was examined in five types of isolated photosystem II D1-D2-cytochrome b 559 reaction center preparations containing either five or six chlorophylls per reaction center. The reaction center complexes were obtained following isolation procedures that differed in chromatographic column material, washing buffer composition and detergent concentration. Two different types of cytochrome b 559 assays were performed. The absolute heme content in each preparation was obtained using the oxidizedminus-reduced difference extinction coefficient of cytochrome b 559 at 559 nm. The relative amount of D1 and cytochrome b 559 a-subunit polypeptide was also calculated for each preparation from immunoblots obtained using antibodies raised against the two polypeptides. The results indicate that the cytochrome b 559 heme content in photosystem II reaction center complexes can vary with the isolation procedure, but the variation of the cytochrome b 559 a-subunit/D1 polypeptide ratio was even greater. This variation was not found in the PSII-enriched membrane fragments used as the RC-isolation starting material, as different batches of membranes obtained from spinach harvested at different seasons of the year or those from sugar beets grown in a chamber under controlled environmental conditions lack variation in their a-subunit/D1 polypeptide ratio. A precise determination of the ratio using an RC1-control sample calibration curve gave a ratio of 1.25 cytochrome b 559 a-subunit per 1.0 D1 polypeptide in photosystem II membranes. We conclude that the variations found in the reaction center preparations were due to the different procedures used to isolate and purify the different reaction center complexes.Keywords: chromatography; cytochrome b 559 ; detergent; immunoblot; photosystem II.Cytochrome (Cyt) b 559 is a hemoprotein component of the photosystem II (PSII) reaction center (RC) complex [1], and it is an integral component of the minimal isolated RC complex still capable of performing primary charge separation. It is composed of two small polypeptides, the a (9 kDa) and b (4.5 kDa) subunits, encoded by the psbE and psbF genes, respectively. Each polypeptide has a single transmembrane a-helical domain [2,3]. The heme iron is bound to a single histidine residue on each subunit [4], and it is located close to the stromal surface of the membrane [2,3,[5][6][7]. However, a location for Cyt b 559 heme on the lumenal side of the PSII membrane has also been proposed, suggesting that two hemes and two copies each of the two subunits are present in the thylakoid membrane [8,9]. Despite numerous studies [8,10,11] A longstanding issue has been the number of Cyt b 559 per PSII complex. Shuvalov and coworkers argued that PSII core complex from spinach with high O 2 -evolution activity contains two Cyt b 559 per PSII [8]. However, the currently accepted value in isolated PSII RCs, based mainly on absorption spectroscopy techniques [1,[23][24][25][26], is one heme per RC. Recent data based on the crystal structure of ...