The mechanisms whereby hormones regulate metabolic processes in cells have been a focus of intensive research for decades. The study of hormonal regulation of hepatic gluconeogenesis and glycolysis has been a particularly interesting area in metabolic regulation because these two opposing processes must be regulated in a concerted fashion in order to permit unidirectional fiux to occur. Regulation of these two processes can be divided for convenience into three categories. The first involves regulation of the supply of substrate. All gluconeogenic substrates as well as glucose reach the liver in subsaturating concentrations. Thus regulation of substrate release into the blood from extrahepatic tissues or provision of substrate from the diet will directly affect hepatic glucose formation and utilization. The second category deals with the very important but relatively slow (hours to days) adaptive changes in enzyme activity due to regulation of gene expression and/or enzyme degradation. The third category is concerned with the minute-to-minute regulation of pathway flux by hormones such as insulin, glucagon, and catecholamines. This report will deal primarily with the third category, with special emphasis on the role of fructose 2, 6-bisphosphate (Fru 2, 6-P,) in this regulation. In addition, adaptive changes in the Fru 2, 6-P2-metabolizing enzyme activities will also be discussed. Although the emphasis will be on the minute-to-minute regulation of glycolysis and gluconeogenesis, it should be clear that regulation in vivo occurs in all three categories simultaneously and in an integrated fashion.