Abstract: Abstract: When the kinetics of interconversion of deoxy[14C]glucose ([14C]DG) and [14C]DG-6-phosphate ([14C]DG-6-P) in brain in vivo are estimated by direct chemical measurement of precursor and products in acid extracts of brain, the predicted rate of product formation exceeds the experimentally measured rate. This discrepancy is due, in part, to the fact that acid extraction regenerates [14C]DG from unidentified labeled metabolites in vitro. In the present study, we have attempted to identify the 14C-labeled compounds in ethanol extracts of brains of rats given [14C]DG. Six 14C-labeled metabolites, in addition to [14C]DG-6-P, were detected and separated. The major acid-labile derivatives, DG-1-phosphate (DG-1-P) and DG-1,6-bisphosphate (DG-1,6-P2), comprised ˜5 and ˜10–15%, respectively, of the total 14C in the brain 45 min after a pulse or square-wave infusion of [14C]DG, and their levels were influenced by tissue glucose concentration. Both of these acid-labile compounds could be synthesized from DG-6-P by phosphoglucomutase in vitro. DG-6-P, DG-1-P, DG-1,6-P2, and ethanol-insoluble compounds were rapidly labeled after a pulse of [14C]DG, whereas there was a 10–30-min lag before there was significant labeling of minor labeled derivatives. During the time when there was net loss of [14C]DG-6-P from the brain (i.e., between 60 and 180 min after the pulse), there was also further metabolism of [14C]DG-6-P into other ethanol-soluble and ethanol-insoluble 14C-labeled compounds. These results demonstrate that DG is more extensively metabolized in rat brain than commonly recognized and that hydrolysis of [14C]DG-1-P can explain the overestimation of the [14C]DG content and underestimation of the metabolite pools of acid extracts of brain. Further metabolism of DG does not interfere with the autoradiographic DG method.