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The development of the ependyma from 6 weeks (wk) gestation to term was studied in 26 human fetuses and infants for immunocytochemical differentiation using antibodies against vimentin, several cytokeratins, glial fibrillary acidic protein (GFAP) and S-100 protein. Acridine orange-RNA fluorescence was uniform in all differentiated ependymal cells. Marked differences were demonstrated among various anticytokeratin antibodies. Vimentin was demonstrated in undifferentiated cells, particularly during mitosis, and persisted as the ependyma matured. It was strong in floor plate cells and processes forming the ventral median septum. Vimentin and cytokeratin CK-904 coexisted with other immunoreactive proteins but disappeared in a caudorstral gradient with maturation. At 8 wk gestation, GFAP was detected in roof plate cells and their processes forming the dorsal median septum. S-100 protein appeared as early as 6 wk and had a more restricted regional distribution than GFAP at all ages. It was strong in the basal plate ependyma of the spinal cord in young fetuses. The temporal and spatial distributions of the immunoreactive proteins studied correlate with evidence that fetal ependymal cells synthesize compounds that attract or repel axonal growth cones to prevent axons from entering the ventricles or deviating from programmed projection pathways. An additional role may be to induce the transformation of radial glial cells in the subventricular zone.