Abstract The formation of 3-phosphoglycerate from ribulose 1, 5-diphosphate, CO2, and H2O by homogeneous preparations of spinach chloroplast ribulose-1,5-P2 carboxylase was investigated at 1 mm bicarbonate—the lowest concentration that sustained a maximal rate of complete photosynthesis by isolated chloroplasts. Under these conditions, the carboxylase was activated by fructose-6-P and deactivated by fructose-1,6-P2, two intermediates of the reductive pentose phosphate cycle. Activation by fructose-6-P was most pronounced at a limiting level of Mg2+ or at neutral or acidic pH. Fructose-6-P induced a shift from sigmoidal to Michaelian kinetics and decreased (up to a factor of 6) the Km for bi-bicarbonate. Fructose-6-P also decreased the Km for Mg2+ and increased the Km for ribulose-1,5-P2. On deactivation by fructose-1,6-P2, the carboxylase showed, in general, its original kinetic characteristics. Certain other intermediates of the reductive pentose phosphate cycle (ribulose-5-P, xylulose-5-P, erythrose-4-P, ribose-5-P) also activated the carboxylase, but less effectively than did fructose-6-P. 6-Phosphogluconate, a compound not formed by chloroplasts, was a strong activator of the enzyme; its mode of action resembled that of fructose-6-P. Fructose-1,6-P2 was the only intermediate of the reductive pentose phosphate cycle that deactivated the carboxylase. Two intermediates in starch synthesis, glucose-1,6-P2 and glucose-6-P, deactivated the carboxylase as well as did fructose-1,6-P2. A regulatory mechanism based on these and other findings is proposed for the conversion of CO2 to starch in photosynthesis.