The significance of the classical subdivision of the retinal primitive neuroepithelium into an outer and an inner neuroblastic layer by the transient fibre layer of Chievitz (LOC) is little understood. We examine here the formation of neuroblastic layers by regenerating fully laminated retinospheroids from dissociated cells of the embryonic chick eye margin in rotary culture. By tracing cellular processes with the fibre-specific F11-antibody in retinospheroids, we occasionally find, in addition to an outer and an inner plexiform layer, a cell-free F11-positive LOC homologue, subdividing the inner nuclear layer. Moreover, we demonstrate that the LOC precisely separates postmitotic AChE-positive cells of the inner retina from an AChE-negative outer part holding all BrdU-labelled mitotic cells. These in vitro data suggest that the inner neuroblastic layer is exclusively composed of AChE-positive cells, thus representing a primary differentiation zone of the retina.