In rat hepatocytes fructose at low concentrations (below 1 mM) stimulated the glycolytic flux as measured by the release of 3H2O from [3-3H]glucose and increased fructose 2,6-bisphosphate [Fru(2,6)P2] levels, without modifying the activity of 6-phosphofructo-2-kinase. Maximal stimulation of the glycolytic pathway by 0.1 mM fructose was observed when hepatocytes were incubated in the presence of physiological concentrations of glucose (8 mM). The rise in Fru(2,6)P2 levels was probably due to an increase in glucose 6-phosphate, which in turn resulted from the stimulation of glucose phosphorylation as measured by the formation of 3H2O from [2-3H]glucose. Furthermore, no effects of low doses of fructose on the glycolytic flux or on glucose phosphorylation were observed in hepatocytes from streptozocin-diabetic rats in which glucokinase is almost absent, or in hepatocytes incubated in the presence of mannoheptulose where glucokinase is inhibited. These results suggest that fructose at low concentrations increases the glycolytic flux by raising Fru(2,6)P2 levels solely as a consequence of the stimulation of glucose phosphorylation.