1. 1. CO 2 fixation of intact spinach chloroplasts is inhibited by nitrite in a pH-dependent mode. At pH 7.3 in the medium 1 mM NaNO 2 and at pH 7.9 5 mM NaNO 2 were required for 50% inhibition. 2. 2. The addition of nitrite leads to an acidification in the stroma. It appears that nitrite renders the envelope permeable for protons resulting in a breakdown of the pH gradient between the external space and the stroma. 3. 3. In view of earlier results on the pH sensitivity of CO 2 fixation it is concluded that this pH shift in the stroma is responsible for the observed inhibition of CO 2 fixation by nitrite. 4. 4. Octanoate and to some extent also high concentrations of bicarbonate and acetate have a similar effect as nitrite in inhibiting CO 2 fixation through an acidification in the stroma. 5. 5. The levels of the intermediates of the CO 2 fixation cycle were measured. A strong rise of the levels of fructose- and sedoheptulose biphosphates and a concomitant decrease of the corresponding monophosphates was observed during inhibition of CO 2 fixation. It appears that the enzymatic steps of the CO 2 fixation cycle responsible for the overall inhibition of CO 2 fixation caused by lowering of the H + concentration in the stroma are fructose- and sedoheptulose bisphosphatase. These two enzymes have an important function in the light regulation of CO 2 fixation.