Ammonium sulfate fractionation of an extract from the leaves of spinach (Spinacia oleracea L.) produced two fractions of phosphofructokinase activity, the first stimulated by inorganic phosphate and the second inhibited by inorganic phosphate. Only the second fraction was obtained from similar treatment of an extract of isolated spinach chloroplasts. The two fractions differed distinctly with respect to kinetics for the substrate fructose 6-phosphate. Evidence for these two types of phosphofructokinase was also obtained with extracts from the leaves of wheat (Triticum aestivum L.), pea (Pisum sativum L.), and maize (Zea mays L.), and the glumes of oat (Avena sativa L.), but not from chive (Allium schoenoprasum L.) leaves, pea cotyledons, or pea roots. It was concluded that most leaves contain phosphofructokinase activity in chloroplasts as well as in the cytoplasm. Spinach chloroplast phosphofructokinase activity, which was at least 2.5 mumoles fructose 1,6-bisphosphate formed per mg chlorophyll per hour, did not result from contamination by cytoplasm or by other cellular organelles, and was not detected until after chloroplasts were broken.