Fructose-6-phosphate 2-kinase and fructose-2,6-bisphosphatase have been partially purified from spinach leaves and their regulatory properties studied. Fructose-6-phosphate 2-kinase was activated by phosphate and fructose 6-phosphate, and inhibited by 3-phosphoglycerate and dihydroxyacetone phosphate. Fructose-2,6-bisphosphatase was inhibited by fructose 6-phosphate and phosphate. The interaction between these effectors was studied when they were varied, alone or in combination, over a range of concentrations representative of those in the cytosol of spinach leaf cells. In conditions when dihydroxyacetone phosphate or 3-phosphoglycerate rise, as is typical during photosynthesis, the fructose 2,6-bisphosphate level will decrease, which will favour sucrose synthesis. In conditions when fructose 6-phosphate accumulates, fructose 2,6-bisphosphate should rise, which will favour a restriction of sucrose synthesis and promotion of starch synthesis.During photosynthesis, triose phosphates are exported from the chloroplasts and converted to sucrose in the cytosol. The first irreversible reaction in the cytosol is catalysed by the cytosolic fructose-l,6-bisphosphatase and Fru(2,6)P is a potent inhibitor of this enzyme [l, 21. In spinach leaves the level of Fru(2,6)P2 decreases when photosynthesis and sucrose synthesis are stimulated by illumination [3] and increases again when sucrose has accumulated [3]. These observations suggest that Fru(2,6)P2 is involved in the regulation of photosynthetic sucrose synthesis but are incomplete because they do not reveal the mechanisms whereby the concentration of Fru(2,6)P2 is being altered. In liver, Fru(2,6)P2 is synthesized and degraded by specific enzymes, the Fru6P 2-kinase and the Fru(2,6)P2 phosphatase [4, 51. Recently it has been shown that similar enzyme activities are present in spinach leaves [6, 71 and that they are modulated by metabolites. In the present article the regulation of Fru6P 2-kinase and Fru(2,6)P2 phosphatase has been studied in more detail, with particular attention being paid to assessing the effect that measured cytosolic concentrations [8 -101 of metabolites would have on the concentration of Fru(2,6)P2.