The steady-state brain/plasma distribution ratios of [14C]deoxyglucose ([14C]DG) for hypoglycemic rats previously determined by measurement of DG concentrations in neutralized acid extracts of freeze-blown brain and plasma exceeded those predicted by simulations of kinetics of the DG model. Overestimation of the true size of the precursor pool of [14C]DG for transport and phosphorylation could arise from sequestration of [14C]DG within brain compartments and/or instability of metabolites of [14C]DG and regeneration of free [14C]DG during the experimental period or extraction procedure. In the present study, the concentrations of [14C]DG and glucose were compared in samples of rat brain and plasma extracted in parallel with perchloric acid or 65% ethanol containing phosphate-buffered saline. The concentrations of both hexoses in acid extracts of brain were higher than those in ethanol, whereas hexose contents of plasma were not dependent on the extraction procedure. The magnitude of overestimation of DG content (about 1.2-to fourfold) varied with glucose level and was highest in extracts isolated from hypoglycemic rats; contamination of the [14C]DG fraction with 14C-labeled nonacidic metabolites also contributed to this overestimation. Glucose concentrations in acid extracts of brain exceeded those of the ethanol extracts by less than 40% for normal and hypoglycemic rats.