In the present investigation we monitored the incorporation of [14C] from [U-14C]glucose into various rat brain glycolytic intermediates of conscious and pentobarbital-anesthetized animals. Labeled glucose was delivered to brain by single bolus intracarotid injection and brain tissue was subsequently prepared at 15, 30, and 45 sec by freeze-blowing. Glycolytic intermediates were then separated by column chromatography. Our results showed a gradual decrease with time of 14C-labeled glucose which gave a calculated rate for glucose metabolism of 0.86 mumol/min/g and 0.56 mumol/min/g in conscious and anesthetized animals, respectively. Compared to the results obtained using conscious animals the administration of pentobarbital not only resulted in a significant attenuation of the rate of glucose metabolism but also caused a similar reduction in the amount of 14C incorporated into several glycolytic intermediates. These intermediates included: glucose 6-phosphate, fructose 6-phosphate, fructose 1,6 diphosphate, dihydroxyacetone phosphate and post glycolytic compounds. In addition, pretreatment with pentobarbital resulted in a 75% increase in the endogenous concentration of glucose, 10% increase in glucose 6-phosphate, no change in fructose 6-phosphate and 42% decrease in lactate compared to levels in brains obtained from conscious animals. These results are discussed in relation to control of glycolysis through coupled regulation at hexokinase-phosphofructokinase.