A fructose-2,6-bisphosphate dephosphorylating enzyme was 3000-fold purified to electrophoretic homogeneity from Saccharomyces cerevisiae. Half-maximal activity was obtained at pH 6.0 with 6 pM fructose 2,6-bisphosphate and 0.15mM Mg2+. On incubation for 90min with fructose 2,6-bisphosphate, about 80% of the substrate appears with an almost linear time dependence as fructose. In the first 30 min a substance accumulates to about 40% of the consumed fructose 2,6-bisphosphate which forms free fructose on mild acid treatment. Formation of fructose 6-phosphate was negligible. The mild-acid-labile intermediate was identified as fructose 2-phosphate by comparative ion-exchange chromatography with authentic fructose 2-phosphate synthesized from fructose l-phosphate [Pontis, H. . The data suggest the reaction sequence fructose 2,6-bisphosphate + fructose 2-phosphate -+ fructose. The designation fructose-2,6-bisphosphate 6-phosphohydrolase is proposed for the enzyme described here.Fructose 2,6-bisphosphate [Fru(2,6)P2] has' been discovered to be a potent activator of 6-phosphofructokinase-1 and inhibitor of fructose-l,6-bisphosphatase in liver, other mammalian tissues and yeast [l, 21. Fru(2,6)P2 is formed in liver by an enzyme catalyzing ATP-dependent phosphorylation of Fru6P in the 2-position. It has been shown that the same enzyme protein also catalyzes dephosphorylation of Fru(2,6)P2 to Fru6P and inorganic phosphate. Modification of the kinase activity to the phosphatase activity of this enzyme is effected by cyclic-AMP-dependent protein phosphorylation. The reverse modification is the result of dephosphorylation of the enzyme [3, 41. In yeast cells a kinase catalyzing phosphorylation of Fru6P to Fru(2,6)P2 has also been described [5, 61. The kinase is activated by cyclic-AMPdependent phosphorylation [5, 71. This is in contrast to liver, where cyclic-AMP-dependent phosphorylation inactivates fructose-6-phosphate kinase-2 and increases fructose-2,6-bisphosphatase activity. In yeast no activity dephosphorylating Fru(2,6)P2 to Fru6P has been detected [5, 71. It is evident from these results that the metabolism of Fru(2,6)P2 in yeast is different from that in mammalian tissues. In the present paper purification of an enzyme to electrophoretic homogeneity from yeast which dephosphorylates Fru(2,6)P2 is described. Fru2P, which has not been described previously as an enzymatic product, and fructose were shown to be the dephosphorylation products.