[6-14C]Glucose is the tracer sometimes recommended to assay cerebral glucose utilization (CMRglc) during transient or brief functional activations, but when used to study visual stimulation and seizures in other laboratories, it underestimated CMRglc. The metabolic fate of [6-14C]glucose during functional activation of cerebral metabolism is not known, and increased labeling of diffusible metabolites might explain underestimation of CMRglc and also reveal trafficking of metabolites. In the current studies cerebral cortex in conscious rats was unilaterally activated metabolically by KCl application, and CMRglc was determined in activated and contralateral control cortex with [6-14C]glucose or 2-[14C]deoxy-glucose ([14C]DG) over a 5- to 7-min interval. Local 14C concentrations were determined by quantitative autoradiography. Labeled precursor and products were measured bilaterally in paired cortical samples from funnel-frozen brains. Left-right differences in 14C contents were small with [6-14C]glucose but strikingly obvious in [14C]DG autoradiographs. CMRglc determined with [6-14C]glucose was slightly increased in activated cortex but 40-80% below values obtained with [14C]DG. [14C]Lactate was a major metabolite of [6-14C]glucose in activated but not control cortex and increased proportionately with unlabeled lactate. These results demonstrate significant loss of labeled products of [6-14C]glucose from metabolically activated brain tissue and indicate that [14C]DG is the preferred tracer even during brief functional activations of brain.