Abstract: Chloroplast FDPase was purified from spinach leaves by ammonium sulfate precipitation, Sephadex G-100 chromatography and DEAE-cellulose chromatography. It was found that treatment of the spinach leaves with liquid nitrogen prior to homoge- nization facilitated the subsequent isolation process, the optimal pH for FDPase activity was 8 to 9 and the enzyme was most stable at pH 6, under which it could be stored over several months without appreciable loss of activity. Acrylamide disc electrophoresis of the final enzyme fraction showed only one essential band. The two forms of FDPase, purified spinach chloroplast FDPase and that in fresilly ruptured spinach chloroplast, behaved differently in some of their kinetic properties. Their activities depended throughout on the concentration of Mg++, but the Km (Mg++) were quite different. The Km (Mg++) of the purified enzyme was about 6.0 mM, that of FDPase in freshly ruptured chloroplasts was, however, 1.0 mM, which corresponded to the concentration of Mg+* in the stroma of illuminated chloroplasts. Mg++ concentration was a limiting factor for the activity of purified FDPase. As the amount of Mg++ in the reaction mixture was lowered, the Km and Vmax were both greatly changed. The shortage of Mg++ could not be compensated by increasing the substrate concentration. The purified FDPase was completely inhibited by 15 μ moles EDTA in the teaction mixture, whereas the FDPase in freshly ruptured chloroplasts was inhibited only 70% by 30 to 45 μ moles EDTA, which was 2 to 3 fold of the concentration sufficient to inhibit completely the activity of the purified enzyme. Moreover, the former was more stable. Its activity did not decline even after incubation for over two hours The FDPase activity was higher in chloroplasts ruptured in 0.2% (w/v) Triton X-100 than that ruptured in water. This phenomenon suggests that this enzyme in vivo might be in some way associated, at least partly; with chloroplast lamellae.