AbstractWe have studied the subcellular fractionation of a clonal line of glial cells from rat brain. In comparison to rat brain, more of the total protein is collected in the crude nuclear fraction following differential centrifugation. When a crude mitochondrial fraction is prepared and resolved on a density gradient, we find that like brain the glial cell crude mitochondrial fraction is also heterogenous. On a sucrose density gradient, such as that used for the preparation of synaptosomes, the glial cell crude mitochondrial fraction resolves into two major fractions. One of these bands sediments to the same isopycnic density as synaptosomes. This fraction contains 20% of the protein, and 50% of the Na‐K ATPase and acid phosphatase of the crude mitochondrial fraction, but virtually no cytochrome oxidase or choline esterase. The fraction consists of numerous membrane structures resembling plasma membranes when examined by electron microscopy. Because the sedimentation properties of the glial membranes are very similar to synaptosomes, we conclude that synaptosomes prepared on sucrose gradients are probably contaminated by these membrane fragments. In contrast to the separation achieved on sucrose gradients, glial membranes separate from synaptosomes on Ficoll‐sucrose gradie