High concentrations of both bicarbonate and formate inhibit photosynthetic O2 evolution at pH 8.0. At this pH, only 2.4% of the total dissolved carbon dioxide exists as CO2. At pH 7.3, where 11% of the total dissolved carbon dioxide exists as CO2, HCO3− no longer inhibits. While formate still inhibits O2 evolution at pH 7.3, its effect can be partially overcome if CO2 is also present. The rate of binding of added 14C-labeled inorganic carbon is nearly 10-fold more rapid when the internal pH of thylakoid membranes is at 6.0 than when it is at 7.8. These observations suggest that CO2, not HCO3−, is initially bound to the photosystem II reaction center and that the location of the binding site is on the inside thylakoid surface. However, additional data presented here suggest that, after binding, CO2 is hydrated to HCO3− + H+ in a pH-dependent reaction. Two possible explanations of the “bicarbonate effect” are presented.