Fructose 2, 6-bisphosphate (Fru-2, 6-P2) was discovered during investigations into the mechanism whereby glucagon, via its second messenger cAMP, stimu lates gluconeogenesis and inhibits glycolysis in liver. It is a potent activator of the glycolytic enzyme 6-phosphofructo-1-kinase and an inhibitor of the gluconeogenic enzyme fructose-1, 6-bisphosphatase, and is thus an important regulatory/signal metabolite that provides a switching mechanism between these two opposing pathways of hepatic carbohydrate metabolism. Fru-2, 6-P2 is synthesized from Fru-6-P (fructose-6-phosphate) and ATP by 6PF-2-K (6-phosphofructose-2-kinase), and is degraded to Fru-6-P and inorganic phos phate by Fru-2, 6-P2ase. These two opposing enzyme reactions are catalyzed by a single unique protein, 6PF-2-KlFru-2, 6-P2ase. It is one of only five known bifunctional enzymes that catalyze opposing reactions, and is unique because it is the only one whose target is a metabolite rather than a protein. The general properties of 6PF-2-KlFru-2, 6-P2ase have been described pre viously in the Annual Review of Biochemistry (I). The present review focuses on the 6PF-2-KlFru-2, 6-P2ase gene family, on hormonal control of 6PF-2-KlFru-2, 6-P2ase gene expression, and on structure-function relationships of both the kinase and bisphosphatase domains and the molecular mechanism (s) of their regulation by phosphorylation. At least five tissue-specific mammalian bifunctional enzyme isoforms, encoded by four different genes, have now been identified and sequenced. The properties and regulation of these enzyme forms as well as two yeast isoforms are reviewed. Recent work on the X-ray crystal structure of the Fru-2, 6-P2ase domain is also summarized. The reader is re ferred elsewhere (1-9) for more comprehensive discussions of the role of Fru-2.6-P2 in the regulation of glycolysis and gluconeogenesis.