Although Mg is abundant within the cell, most is chelated to various organic molecules; only a small fraction of intracellular Mg is in the free (ionized) form, Mgtt Free Mg²⁺ regulates manyenzyme activities in cells. The effect of free Mg²⁺ concentration on the activities of spinach hloroplast fructose-1, 6-bisphosphatase (FBPase) and ribulose 1, 5-bisphosphate carboxylase (rubisco) was examined. Free Mg²⁺ concentrations in the assay mixtures were directly measured by a Mgtsensitive dye, mag-fura-2. FBPase was activated by a physiological concentration range of free Mgt, but the activation of rubisco was not observed. These results suggest that in illuminated chloroplasts, the increase in free Mg²⁺ activates FBPase, and this may be a physiological factor to stimulate CO₂ fixation.