Abstract All neurons and macroglial cells of vertebrates derive from the neuroepithelium. Neuroepithelial (NE) cells first proliferate and, after closure of the neural tube, some cells start generating neurons. It is still unclear what triggers differentiation but apparently there is interplay between extrinsic (secreted or transmembrane signals) and intrinsic factors. Diriving from the embryonic ectoderm, the NE cells inherit epithelial characteristics. It has been shown in other developmental systems that epithelial determinants, such as cell-cell contacts and contact to basal laminar components can guide differentiation. The key epithelial features include cell polarity, and tight junctions. We studied these in the NE at two developmental stages, the neural plate, a proliferative stage and the neural tube, a differentiative stage. The polarity of membrane proteins in NE cells was studied with polarly budding viruses. Mouse embryos were infected with Fowl plague- and vesicular stomatitis viruses and cultured in a whole embryo culture system. Viral envelope proteins (HA and G-protein) were localized by indirect immunofluorescence and immunoelectron microscopy. HA was polarized in the plate stage neuroepithelial cells, whereas in the tube it was not polarized anymore. It is also shown by penetrance of apically injected horseradish peroxidase that in the neural plate, NE cells have functional tight junctions. At this stage, they also express occludin, a transmembrane protein of tight junctions, as shown by indirect immunofluorescence. In the neural tube, the paracellular barrier is lost and there is no occludin expression. In contrast, expression of ZO-1, a cytoplasmic protein binding to occiudin, is upregulated. The downregulation of these epithelial features occurs in all NE cells, irrespective of their mode of division and before any neurons are generated in the NE. The change is initiated already at the plate stage and coincides with the switch from E- to N-cadherin. Later, with birth of neurons, the proliferative cell layer also looses contact to basal lamina. This is probably an important step in the regulation of neurogenesis. Furthermore, lack of apico-basolateral polarity of non-anchored membrane proteins may contribute to the mechanism of rapid neuron generation. Until now, it has been impossible to distinguish a neuroepithelial cell preparing for neuron generation from the surrounding cells that give rise to two precursor cells. In this study, the immediate neuron precursors are shown to express the antiproliferative gene TIS2 1. Using this new marker and ISH in serial sections, we show that the switch to differentiation is initiated in single NE cells.