AbstractAnin vitroslice culture was established for investigating olfactory neural development. The olfactory epithelium was dissected from embryonic day 13 rats; 400μm slices were cultured for 5 days in serum‐free medium on Millicell‐CM membranes coated with different substrates. The slices were grown in the absence of their appropriate target, the olfactory bulb, or CNS derived glia. The cultures mimic many features ofin vivodevelopment. Cells in the olfactory epithelium slices differentiate into neurons that express olfactory marker protein (OMP). OMP‐positive cells have the characteristic morphology of olfactory receptor neurons: a short dendrite and a single thin axon. The slices support robust axon outgrowth. In single‐label experiments, many axons expressed neural specific tubulin, growth‐associated protein 43 and OMP. Axons appeared to grow equally well on membranes coated with type I rat tail collagen, laminin or fibronectin. The cultures exhibit organotypic polarity with an apical side rich in olfactory neurons and a basal side supporting axon outgrowth. Numerous cells migrate out of the slices, of which a small minority was identified as neurons based on the expression of neural specific tubulin and HuD, a nuclear antigen, expressed exclusively in differentiated neurons. Most of the migrating cells, however, were positive for glial fibrilary acidic protein and S‐100, indicating that they are differentiated glia. A subpopulation of these glial cells also expressed low‐affinity nerve growth factor receptors, indicating that they are olfactory Schwann cells. Both migrating neurons and glia were frequently associated with axons growing out of the slice. In some cases, axons extended in advance of migrating cells. This suggests that olfactory receptor neurons in organotypic cultures require neither a pre‐established glial/neuronal cellular terrain nor any target tissue for successful axon outgrowth. Organotypic olfactory epithelial slice cultures may be useful for investigating cellular and molecular mechanisms that regulate early olfactory development and function.