Cytochrome b-559 is an integral component of photosystem II complexes from both plants and cyanobacteria. However, the number of cytochrome b-559 associated with the photosystem II reaction center has been the subject of controversy. Some studies have concluded that there is one heme equivalent of cytochrome b-559 per reaction center, some studies have found two, and some studies have reported intermediate values. Most of the previous experiments have used only one method to quantitate the antenna size of the preparation. In this study, we compare the cytochrome b-559 content in a cyanobacterial and a plant photosystem II preparation. The plant preparation is derived from spinach, and previous work has shown that it has an antenna size of approximately 100 chlorophylls [MacDonald, G. M., & Barry, B. A. (1992) Biochemistry 31, 9848-9856]. The cyanobacterial preparation is from Synechocystis sp. PCC 6803, and previous work has shown that it has an antenna size of approximately 60 chlorophylls [Noren, G. H., Boerner, R. J., & Barry, B. A. (1991) Biochemistry 30, 3943-3950]. Both preparations are isolated through the use of ion-exchange chromatography, and both preparations are monodisperse in the same nonionic detergent. In our comparative study, we quantitate antenna size by three different methods. Our work shows that, depending on the method used to estimate antenna size, the oxygen-evolving spinach photosystem II preparation contains 0.82-1.0 cytochrome b-559 per reaction center, while the oxygen-evolving cyanobacterial preparation contains 1.5-2.1 cytochrome b-559 per reaction center.