We have developed a serum-free chemically defined medium which allows the obtainment of a primary culture highly enriched in ciliated ependymal cells. Serum was never used. Mechanically dissociated neonatal rat brain hemisphere cells were seeded on a fibronectin substratum. Culture medium was minimum Eagle's medium until day 14 in vitro and Waymouth's MD 705/l medium thereafter. Both media were supplemented with insulin (5 micrograms/ml), transferrin (10 micrograms/ml) and fatty acid-free bovine serum albumin (0.5 mg/ml). In these conditions, without addition of growth factors, the culture was enriched in ependymal cells; with the addition of thrombin cell growth was stimulated and moreover a nearly pure culture of ependymal cells was obtained. In this very simple culture medium, cells looked healthy up to two months after seeding and about 75% of the cells were ciliated. In a fraction of these cells the glial fibrillary acidic protein, a protein specific for astroglial cells in the central nervous system, was immunohistochemically revealed. The availability of an almost pure primary culture of ependymal cells will make many studies possible on this cell type, particularly studies on the development of these cells, on the regulation of their genetic expression and on their physiological function.