The rates of [6-'4C]-glucose oxidation by reconstituted systems of cytosol and mitochondria or cytosol and synaptosomes were essentially the same as the rate of oxidation of [3-'4C]-3-hydroxybutyrate. However, the rate of [U-'4C]-glutamine oxidation by mitochondria was 2.5 times that by synaptosomes. The addition of glutamine (5 mM) caused a reduction in the rates of oxidation of [6-'4C]-glucose of 20 % and 40 % by mitochondria and synaptosomes, respectively. Conversely, the addition of glucose (5 mM) had little or no effect on the rate of [U-I4C]glutamine oxidation by either organelle. Amino-oxyacetate decreased [U-'4C]glutamine oxidation by mitochondria more than 35 % but had little or no effect on the rate of glutamine oxidation by synaptosomes. When glucose ( 5 mM) was added to [3-'4C]-3-hydroxybutyrate, the rates of oxidation by the mitochondria and synaptosomes were increased by 65 % and 77 % , respectively. However, in the reverse situation the addition of 3-hydroxybutyrate decreased [6-L4C]-glucose oxidation by synaptosomes (35%) but did not decrease the rate by mitochondria. These results suggest that differences in the rates of substrate utilization by mitochondria and synaptosomes and differences in substrate interactions in these two subcellular organelles may contribute to metabolic compartmentation in the brain.