In this work the feasibility of measuring neuronal-glial metabolism in rat brain in vivo using co-infusion of [1,6-(13)C(2)]glucose and [1,2-(13)C(2)]acetate was investigated. Time courses of (13)C spectra were measured in vivo while infusing both (13)C-labeled substrates simultaneously. Individual (13)C isotopomers (singlets and multiplets observed in (13)C spectra) were quantified automatically using LCModel. The distinct (13)C spectral pattern observed in glutamate and glutamine directly reflected the fact that glucose was metabolized primarily in the neuronal compartment and acetate in the glial compartment. Time courses of concentration of singly and multiply-labeled isotopomers of glutamate and glutamine were obtained with a temporal resolution of 11 min. Although dynamic metabolic modeling of these (13)C isotopomer data will require further work and is not reported here, we expect that these new data will allow more precise determination of metabolic rates as is currently possible when using either glucose or acetate as the sole (13)C-labeled substrate.