Fructose loading results in hepatic accumulation of fructose 1-phosphate (Fru1 P). The goals of the present experiments were: first, to distinguish between ATP, intracellular inorganic phosphate (Pi), and extracellular Pi as sources of phosphate for the phosphorylation of fructose, and second, to examine the influence of ATP and Fru1 P on movement of phosphate into and out of these three pools. To achieve these goals, 31P-NMR was used to monitor the response of hepatic ATP, Pi and Fru1 P to two consecutive injections of fructose. The first was administered with ATP at the control level, and the second, 1 h after the first, with ATP at 65% of the control level. Changes in intra- and extracellular Pi were distinguished by correlating measurements of total NMR-detectable phosphorus and NMR-detectable Pi with measurements of plasma Pi. The initial fructose injection resulted in rapid accumulation of Fru1 P, small decreases in plasma and NMR-detectable Pi and a dramatic decrease in ATP. Total NMR-detectable phosphorus did not change, suggesting that phosphate did not enter or leave the liver. Therefore, accumulation of Fru1 P was initially balanced by an equivalent decrease in ATP, without large changes in Pi. Following the second injection, when ATP was at 65% of control. Fru1 P accumulated at approximately the same rate and to the same level as achieved following the first injection. There was little further change in ATP and a marked decrease in NMR-detectable Pi, while plasma Pi was higher than after the first injection. Therefore the greater decrease in NMR-detectable Pi following the second injection represented a significant decrease in intracellular Pi. Return of Fru1 P to control coincided with a dramatic increase in plasma Pi, and a decrease in total NMR-detectable phosphate. This suggests that phosphate released from Fru1 P entered the extracellular space. These data suggest the mechanisms by which intracellular Pi is regulated. When sufficient ATP is available, ATP hydrolysis supplies phosphate for the synthesis of Fru1 P, and prevents a significant decrease in intracellular Pi. When ATP is reduced, accumulation of Fru1 P depletes intracellular Pi. Therefore, decreased availability of ATP correlates with increased utilization of intracellular Pi. When Fru1 P returns to control, the increase in intracellular Pi is limited by release of Pi into the plasma.