Neuron, astrocyte, and oligodendrocyte cultures which were established from developing rat brain were examined for their utilization of glucose, ketone bodies, and free fatty acids by oxidative processes. 14CO2 production was measured in these cells from [1-14C] or [6-14C]glucose; [1-14C]octanoate and [1-14C], [6-14C], or [16-14C]palmitate; and [3-14C]acetoacetate and D(-)-3-hydroxy[3-14C]butyrate. Pyruvate dehydrogenase (EC 1.2.4.1.) and 3-oxoacid-CoA transferase (EC 2.8.3.5) activities were found at high levels in each of the cell populations. Astrocytes and oligodendrocytes produced much more 14CO2 from [1-14C]glucose than from [6-14C]glucose, indicating substantial hexose monophosphate shunt activity. This process was not as active in neurons. All three cell populations readily utilized the ketone bodies for oxidative metabolism at rates 7-9 times greater than they utilized glucose. Only astrocytes were able to utilize fatty acids for 14CO2 production, and the rate of utilization was greater than that of the ketone bodies. We found that the metabolic patterns of these brain cells which were derived from the developing brain complement the nature of the diet of the suckling animal which is rich in fat and low in carbohydrate. They readily utilized the ketone bodies or fatty acids and spared glucose for processes that metabolites of fat cannot fulfill.