Neuroepithelial and radial glial cells span between the ventricular and the pial surfaces of the neural tube and express two intermediate filaments (IFs), nestin and vimentin, which form a filamentous network throughout the length of the cells. In this report we study the polymerization characteristics of nestin and examine how mutations affect the assembly and localization of the nestin protein in cultured cells and in the developing CNS of transgenic mice. A wild-type rat nestin gene transfected into the IF-free SW13 cell line failed to assemble into a filamentous network but was incorporated into the existing IF network of a subclone expressing vimentin, demonstrating that nestin requires vimentin for proper assembly. In transgenic mice, rat nestin formed a network indistinguishable from that formed by endogenous nestin and vimentin, but a mutant form lacking five amino acids at the carboxy terminus of the rod domain was largely restricted to the pial endfeet. Since nestin mRNA is localized to the pial endfoot region we propose that both transgenes are translated there, but that the wild-type protein is preferentially incorporated into the IF network. These observations provide evidence for hierarchical assembly and a complex organization of the IF network along the ventricular-pial axis in the early CNS.