Labeled glucose and its analogs are widely used in imaging and metabolic studies of brain function, astrocyte–neuron interactions, and neurotransmission. Metabolite shuttling among astrocytes and neurons is essential for cell–cell transfer of neurotransmitter precursors and supply and elimination of energy metabolites, but dispersion and release of labeled compounds from activated tissue would reduce signal registration in metabolic labeling studies, causing underestimation of focal functional activation. Processes and pathways involved in metabolite trafficking and release were therefore assessed in the auditory pathway of conscious rats. Unilateral monotonic stimulation increased glucose utilization (CMRglc) in tonotopic bands in the activated inferior colliculus by 35–85% compared with contralateral tissue when assayed with [14C]deoxyglucose (DG), whereas only 20–30% increases were registered with [1- or 6-14C]glucose. Tonotopic bands were not evident with [1-14C]glucose unless assayed during halothane anesthesia or pretreatment with probenecid but were detectable with [6-14C]glucose. Extracellular lactate levels transiently doubled during acoustic stimulation, so metabolite spreading was assessed by microinfusion of [14C]tracers into the inferior colliculus. The volume of tissue labeled by [1-14C]glucose exceeded that by [14C]DG by 3.2- and 1.4-fold during rest and acoustic activation, respectively. During activation, the tissue volume labeled by U-14C-labeled glutamine and lactate rose, whereas that by glucose fell 50% and that by DG was unchanged. Dispersion of [1-14C]glucose and its metabolites during rest was also reduced 50% by preinfusion of gap junction blockers. To summarize, during brain activation focal CMRglc is underestimated with labeled glucose because of decarboxylation reactions, spreading within tissue and via the astrocyte syncytium, and release from activated tissue. These findings help explain the fall in CMRO2/CMRglc during brain activation and suggest that lactate and other nonoxidized metabolites of glucose are quickly shuttled away from sites of functional activation. © 2007 Wiley-Liss, Inc.