The rate of incorporation of carbon from [1-13C]glucose into the [4-CH2] and [3-CH2] of cerebral glutamate was measured in the rat brain in vivo by 1H-observed, 13C-edited (POCE) nuclear magnetic resonance (NMR) spectroscopy. Spectra were acquired every 98 s during a 60-min infusion of [1-13C]glucose. Complete time courses were obtained from six animals. The measured intensity of the unresolved [4-13CH2] resonances of glutamate and glutamine increased exponentially during the infusion and attained a steady state in approximately 20 min with a first-order rate constant of 0.130 +/- 0.010 min-1 (t1/2 = 5.3 +/- 0.5 min). The appearance of the [3-13CH2] resonance in the POCE difference spectrum lagged behind that of the [4-13CH2] resonance and had not reached steady state at the end of the 60-min infusion (t1/2 = 26.6 +/- 4.1 min). The increase observed in 13C-labeled glutamate represented isotopic enrichment and was not due to a change in the total glutamate concentration. The glucose infusion did not affect the levels of high-energy phosphates or intracellular pH as determined by 31P NMR spectroscopy. Since glucose carbon is incorporated into glutamate by rapid exchange with the tricarboxylic acid (TCA) cycle intermediate alpha-ketoglutarate, the rate of glutamate labeling provided an estimate of TCA cycle flux. We have determined the flux of carbon through the TCA cycle to be approximately 1.4 mumols g-1 min-1. These experiments demonstrate the feasibility of measuring metabolic fluxes in vivo using 13C-labeled glucose and the technique of 1H-observed, 13C-decoupled NMR spectroscopy.