Prereduction of the intersystem pool in isolated spinach chloroplasts leads to the appearance of a slow phase in the field-indicating 515-nm change induced by a flash. Measurements with the pH-indicator dye cresol red show that a proton uptake is associated with this slow 515-nm change. When water is the electron donor to photosystem II, electron transfer to ferricyanide is associated with the uptake of more than one proton per electron. Tetraphenylboron upon oxidation by system II releases a proton directly into the medium; yet, flash-induced electron transport from tetraphenylboron to ferricyanide is accompanied by a net uptake of protons from the medium. The above four results demonstrate the existence of two proton translocation sites in the chain between the two photoacts; the first two observations locate the new site at the oxidizing side of the plastoquinone pool.