The pyruvate dehydrogenase complex (PDC), required for complete glucose oxidation, is essential for brain development. Although PDC deficiency is associated with a severe clinical syndrome, little is known about its effects on either substrate oxidation or synthesis of key metabolites such as glutamate and glutamine. Computational simulations of brain metabolism indicated that a 25% reduction in flux through PDC and a corresponding increase in flux from an alternative source of acetyl-CoA would substantially alter the 13 C NMR spectrum obtained from brain tissue. Therefore, we evaluated metabolism of [1,6- 13 C 2 ]glucose (oxidized by both neurons and glia) and [1,2- 13 C 2 ]acetate (an energy source that bypasses PDC) in the cerebral cortex of adult mice mildly and selectively deficient in brain PDC activity, a viable model that recapitulates the human disorder. Intravenous infusions were performed in conscious mice and extracts of brain tissue were studied by 13 C NMR. We hypothesized that mice deficient in PDC must increase the proportion of energy derived from acetate metabolism in the brain. Unexpectedly, the distribution of 13 C in glutamate and glutamine, a measure of the relative flux of acetate and glucose into the citric acid cycle, was not altered. The 13 C labeling pattern in glutamate differed significantly from glutamine, indicating preferential oxidation of [1,2- 13 C]acetate relative to [1,6- 13 C]glucose by a readily discernible metabolic domain of the brain of both normal and mutant mice, presumably glia. These findings illustrate that metabolic compartmentation is preserved in the PDC-deficient cerebral cortex, probably reflecting intact neuron–glia metabolic interactions, and that a reduction in brain PDC activity sufficient to induce cerebral dysgenesis during development does not appreciably disrupt energy metabolism in the mature brain.