In order to investigate the effect of fructose on the metabolic rates of liver glycolysis, changes in fructose 2,6-bisphosphate content and fructose 6-phosphate, 2-kinase (F6P2kinase) activity were examined using perfused rat liver. When the starved liver was perfused with 0.5 mM fructose, the fructose 2,6-bisphosphate level was 6.66 nmol/g liver, or the maximum. However, with further increases in the fructose concentration, increments in the fructose 2,6-bisphosphate level gradually decreased. F6P2kinase was inactive in the starved rat liver. By perfusion with the medium containing fructose or even without fructose, however, the enzyme was activated. The suppression of the increase in the fructose 2,6-bisphosphate level by perfusion at higher fructose concentrations was at least partly due to the inhibition of F6P2kinase by F1P.