The generally accepted metabolic concept that fructose 2,6-bisphosphate (Fru-2,6-P 2 ) inhibits gluconeogenesis by directly inhibiting fructose 1,6-bisphosphatase is based entirely on in vitro observations. To establish whether gluconeogenesis is indeed inhibited by Fru-2,6-P 2 in intact animals, a novel NMR method was developed using [U-13 C]glucose and 2 H 2 O as tracers. The method was used to estimate the sources of plasma glucose from gastric absorption of oral [U-13 C]glucose, from gluconeogenesis, and from glycogen in 24-h fasted rats. Liver Fru-2,6-P 2 increased Ïł10-fold shortly after the glucose load, reached a maximum at 60 min, and then dropped to base-line levels by 150 min. The gastric contribution to plasma glucose reached Ïł50% at 30 min after the glucose load and gradually decreased thereafter. Although the contribution of glycogen to plasma glucose was small, glucose formed from gluconeogenesis was substantial throughout the study period even when liver Fru-2,6-P 2 was high. Liver glycogen repletion was also brisk throughout the study period, reaching Ïł30 mol/g at 3 h. These data demonstrate that Fru-2,6-P 2 does not inhibit gluconeogenesis significantly in vivo.Plasma glucose is preserved by gluconeogenesis after exhaustion of glycogen stores during a moderate fast. Following an oral glucose load, gluconeogenesis is thought to be modulated by allosteric regulation of fructose-1,6-bisphosphatase (1). This is an eminently satisfying model because a key regulatory site in glycolysis and gluconeogenesis occurs at level of fructose-6-P and fructose-1,6-P 2 (Fig. 1). Phosphofructokinase-1, the glycolytic enzyme, is potently activated by fructose-2,6-P 2 , whereas fructose-1,6-bisphosphatase, the gluconeogenic enzyme, is thought to be inhibited by this same effector molecule (2, 3). Thus, by regulating the activities of phosphofructo-1-kinase and fructose-1,6-bisphosphatase in a reciprocal manner, Fru-2,6-P 2 is thought to serve as an elegant regulator of glucose usage/production by the liver after an oral glucose load. This generally accepted model is based on kinetic analysis of fructose-1,6-bisphosphatase in vitro, which shows that Fru-2,6-P 2 competes with Fru-1,6-P 2 for the active site of fructose-1,6-bisphosphatase and that both molecules have similar affinity constants, 1-5 M (4 -6). However, the in vivo concentrations of Fru-1,6-P 2 in fasted and fed livers are 20 and 35 M, respectively, whereas those of Fru-2,6-P 2 are 1 and 8 M, respectively (7-9). This suggests that it would be difficult for Fru-2,6-P 2 to have a significant direct effect on fructose-1,6-bisphosphatase activity in vivo based simply upon concentration differences. Some evidence has been presented that suggests Fru-2,6-P 2 is not a potent inhibitor of gluconeogenesis in intact animals. For example, Kuwajima et al. (10) reported continual production of liver glycogen in sucrose-fed rats despite high levels of Fru-2,6-P 2 , and Hue and Bartrons (11) observed stimulated glucose production by glucagon in isolated hepatocytes reg...