In rat hepatocytes, vanadate increases fructose 2,6-bisphosphate (Fru-2,6-P2) in a time- and dose-dependent manner, and counteracts the decrease in this metabolite caused by glucagon, forskolin or exogenous cyclic AMP. Vanadate does not directly modify the activity of 6-phosphofructo-2-kinase, even though it can counteract the inactivation of this enzyme caused by glucagon. Furthermore, vanadate raises the yield of 3H2O from [3-3H]glucose, indicating that it increases the flux through 6-phosphofructo-1-kinase. Moreover, vanadate in hepatocytes incubated in the presence of glucose increases the production of both lactate and CO2. Therefore vanadate has insulin-like effects on the glycolytic pathway in rat hepatocytes. These results clearly contrast with our previous observation that vanadate exerts glycogenolytic non-insulin-like effects on glycogen synthase and phosphorylase.