It is shown that restoration of photoinduced electron flow and O 2 evolution with Mn 2+ in Mn-depleted photosystem II (PSII) membrane fragments isolated from spinach chloroplasts is considerably increased with bicarbonate in the region pH 5.0–8.0 in bicarbonate-depleted medium. In buffered solutions equilibrated with the atmosphere (nondepleted of bicarbonate), the bicarbonate effect is observed only at pH lower than the pK of H 2 CO 3 dissociation (6.4), which indicates that HCO 3 − is the essential species for the restoration effect. The addition of just 2 Mn 2+ atoms per one PSII reaction center is enough for the maximal reactivation when bicarbonate is present in the medium. Analysis of bicarbonate concentration dependence of the restoration effect reveals two binding sites for bicarbonate with apparent dissociation constant ( K d ) of ≈2.5 μM and 20–34 μM when 2,6-dichloro- p -benzoquinone is used as electron acceptor, while in the presence of silicomolybdate only the latter one remains. Similar bicarbonate concentration dependence of O 2 evolution was obtained in untreated Mn-containing PSII membrane fragments. It is suggested that the K d of 20–34 μM is associated with the donor side of PSII while the location of the lower K d binding site is not quite clear. The conclusion is made that bicarbonate is an essential constituent of the water-oxidizing complex of PSII, important for its assembly and maintenance in the functionally active state.