Addition of sorbitol, which facilitated reductions in reaction medium osmotic potential from standard (0.33 molar sorbitol, -10 bars) isotonic conditions to a stress level of 0.67 molar sorbitol (-20 bars), inhibited the photosynthetic capacity of isolated spinach (Spinacia oleracea) chloroplasts. This inhibition, which ranged from 64 to 74% under otherwise standard reaction conditions, was dependent on reaction medium inorganic phosphate concentration, with the phosphate optimum for photosynthesis reduced to 0.05 millimolar at the low osmotic potential stress treatment from a value of 0.25 millimolar under control conditions.Stromal alkalating agents such as NH(4)Cl (0.75 millimolar) and KCl (35 millimolar) were also found to affect the degree of low osmotic potential inhibition of photosynthesis. Both agents doubled the rate of NaHCO(3)-supported O(2) evolution under the stress treatment, while hardly affecting the control rate at optimal concentrations. These agents also reduced the length of the lag phase of photosynthetic O(2) evolution under the stress treatment to a much greater degree. The rate-enhancement effect of these agents under the stress treatment was reversed by sodium acetate, which is known to facilitate stromal acidification.The reaction medium pH optimum for photosynthesis under the stress treatment was higher than under control conditions. In the presence of optimal NH(4)Cl, this shift was no longer evident.Internal pH measurements indicated that the stress treatment caused a 0.43 and 0.24 unit reduction in the stromal and intrathylakoid pH, respectively, under illumination. This osmotically induced acidification was not evident in the dark. The presence of 0.75 millimolar NH(4)Cl partially reversed the osmotically induced reduction in the illuminated stromal pH. It was concluded that stromal acidification is a mediating mechanism of the most severe site of low osmotic potential inhibition of the photosynthetic process.