[1-¹³C]Glucose, [2-¹³C]acetate and [3-¹³C]lactate were infused into male Sprague–Dawley rats (150–170 g) for periods of 3–100 min (n=4 per time) and neocortex extracts were analyzed using ¹³C-edited ¹H magnetic resonance (MR) spectroscopy. The time dependence of the [4-¹³C]glutamine/[4-¹³C]glutamate labeling ratio was significantly different for all three substrates infused (p<0.001) and showed that acetate is primarily utilized by glia and lactate by neurons, whereas glucose is ubiquitous. The ratio of second- to first-turn TCA cycle labeling for glutamine was significantly lower for acetate (30–100 min infusion; p<0.02) and greater for lactate (10–30 min; p<0.02) than for glucose infusions, while the C-2/C-4 glutamate labeling ratio was similar for all the three substrates. This indicated that transfer of [2-¹³C]acetate-derived [4-¹³C]glutamine to neurons was preferred to reentry of label into the glial TCA cycle and that the neuronal TCA cycle turnover is significantly faster than that for glia. Fitting parameters of a function representing a pseudo-first-order process to the time dependence of labeling demonstrated that GABA labeling reaches steady state faster with glutamine labeled from [2-¹³C]acetate than with glutamate labeled from [3-¹³C]lactate. It is concluded that lactate represents a significant improvement over glucose in the study of neuronal metabolism and complements the use of acetate to study glial metabolism and glial/neuronal metabolic relationships.