Reaggregate cultures have been formed from cell suspensions of neonatal rat retinas. Histological sections of the reaggregates showed evidence of lamination with central rosettes formed around a lumen, a clear neuropil layer and an outer cellular layer. Each of the major retinal cell types, except ganglion cells, could be positively identified using cell type-specific antibodies to label cryostat sections. Many of these were found to occupy positions within the reaggregates similar to those found in the intact retina. Electron microscopic observations showed abundant immature and mature synaptic endings within the neuropil layer, including a number of ribbon synapses. Examination of the rosettes showed an arrangement of Müller glia and photoreceptors that closely resembled that of the intact retina. Within the lumen of rosettes, photoreceptors were found to contain stacks of disc-like membranes bounded by a plasma membrane, analogous to immature outer segments. The photoreceptors within rosettes also underwent molecular differentiation and expressed an outer segment specific marker. The findings suggest that retinal cells have intrinsic properties that allow them to organize themselves into a correctly laminated structure and that particular cell interactions are necessary for continued differentiation of at least rod photoreceptors and Müller cells.