Neurons have a high demand for cholesterol to develop and maintain membrane-rich structures like axons, dendrites and synapses, but it remains unclear, whether they can satisfy their need by costly de novo synthesis. To address this, we compared cholesterol synthesis in serum-free cultures of highly purified CNS neurons and glial cells from postnatal rats. We observed marked cell-specific differences: Compared with glial cells, neurons showed different profiles of biosynthetic enzymes, post-squalene precursors and cholesterol metabolites, and they produced cholesterol less efficiently, possibly because of very low levels of lanosterol-converting enzymes. Astrocytes responded to inhibition of cholesterol synthesis with a much stronger up-regulation of biosynthetic enzymes than neurons. Our results support the idea that neurons cannot produce cholesterol efficiently and that they depend on an external source of this lipid.