The oxidation of [1,3-14C] glycerol to 14CO was measured in slices, whole homogenates, and subcellular fractions of rat brain. In all of these tissue preparations, the Lineweaver-Burk plots of glycerol oxidation were biphasic, yielding two apparent Km and V values. Similar kinetic characteristics were obtained with brain homogenates from guinea pig, mouse, rabbit, monkey, and pig. In other tissues of the rat, including heart, kidney, liver, and skeletal muscle, the Lineweaver-Burk plots for glycerol oxidation were not biphasic but were linear. Heating the brain homogenates for five minutes at 5 degrees C caused a 50% decrease in the rate of oxidation of glycerol without a change in the biphasic double reciprocal plot. The addition of purified glycerol kinase (EC 2.7.1.30) to the homogenate caused an increase in the rate of oxidation and resulted in linear Lineweaver-Burke plot. Brain mitochondria were prepared by two different methods, both of which yielded an enrichment of glycerol oxidation. In contrast, the rate of glucose oxidation was higher in homogenates than in mitochondria, and glucose competed with glycerol as substrate only extramitochondrially. The effects of various metabolic inhibitors suggested the participation of intact, coupled mitochondria, of glycolytic enzymes, and of electron transport in the oxidation of glycerol. The data support the primary localization of glycerol oxidation in nonsynaptic mitochondria in brain and the presence in that organelle of enzymes of the Embden-Meyerhoff pathway or an as yet unidentified system for oxidizing this compound.