In this study, the reversible conversion between the high- (HP) and low-potential (LP) forms of Cytb(559) has been analyzed in Tris-washed photosystem II (PSII) enriched membranes. These samples are deprived of the Mn cluster of the water-oxidizing complex (WOC) and the extrinsic regulatory proteins. The results obtained by application of optical and EPR spectroscopy reveal that (i) under aerobic conditions, the vast majority of Cytb(559) exhibits a low midpoint potential, (ii) after removal of O(2) in the dark, a fraction of Cytb(559) is converted to the high-potential form which reaches level of about 25% of the total Cytb(559), (iii) a similar dark transformation of LP --> HP Cytb(559) occurs under reducing conditions (8 mM hydroquinone), (iv) under anaerobic conditions and in the presence of 8 mM hydroquinone, about 60% of the Cytb(559) attains the HP form, (v) the interconversion is reversible with the re-establishment of aerobic conditions, and (vi) aerobic and oxidizing conditions (2 mM ferricyanide or 0.5 mM potassium iridate) induce a decrease of the amount of the HP form, also showing that the conversion is reversible. This reversible interconversion between LP and HP Cytb(559) is not observed in PSII membrane fragments with an intact WOC. On the basis of these findings, the possibility is discussed that the O(2)-dependent conversion of Cytb(559) in PSII complexes lacking a functionally competent WOC is related to a protective role of Cytb(559) in photoinhibition and/or that it is involved in the regulation of the assembly of a competent water-oxidizing complex in PSII.