AbstractIn order to analyze intercellular communication between ependymal cells in mammalian brain, we have studied gap junctional communication of ependymal and glial cells in long term primary cultures derived from fetal mouse or rat hypothalamus and choroid plexus obtained in serum supplemented media with two complementary methods: (1) dye transfer of Lucifer Yellow CH after intracellular microinjection of the different cellular types, and (2) freeze‐fracture of the same cultured ependymal cells. In our culture conditions, we have shown that the GJIC capacity to transfer dye was very different according to cellular types microinjected with Lucifer Yellow CH in the following respects: (1) in ependymal cells, GJIC was always important: ciliated ependymal cells, which are numerous in hypothalamic ependymal cultures (10–120 coupled cells), choroidal ependymocytes in plexus cultures (15–250 coupled cells), and non‐choroidal ependymocytes in diencephalic roof cultures (10–30 coupled cells), and (2) in astroglial cells found in these primary cultures, no GJIC was observed in spite of the presence of well‐differentiated gap junctions revealed by freeze‐fracture replicas. All these results show a strong GJIC in ependymal cells and indicate the very good functional state of these c