The mammalian brain relies primarily on glucose as a fuel to meet its high metabolic demand. Among the various techniques used to study cerebral metabolism, C-13 magnetic resonance spectroscopy (MRS) allows following the fate of C-13-enriched substrates through metabolic pathways. We herein demonstrate that it is possible to measure cerebral glucose metabolism in vivo with sub-second time resolution using hyperpolarized C-13 MRS. In particular, the dynamic C-13-labeling of pyruvate and lactate formed from C-13-glucose was observed in real time. An ad-hoc synthesis to produce [2,3,4,6,6-H-2(5), 3,4-C-13(2)]-D-glucose was developed to improve the 13C signal-to-noise ratio as compared to experiments performed following [U-H-2(7), U-C-13]-D-glucose injections. The main advantage of only labeling C3 and C4 positions is the absence of C-13-C-13 coupling in all downstream metabolic products after glucose is split into 3-carbon intermediates by aldolase. This unique method allows direct detection of glycolysis in vivo in the healthy brain in a noninvasive manner.