Olfactory neurons have the rare property of being replaced throughout life. Factors regulating different developmental stages of olfactory receptor neurons (ORNs) are of great interest, because such factors might be used to extend regeneration in the post-developmental brain and spinal cord. Also, these factors may potentially be exploited to treat various smell disorders arising from changes in the olfactory epithelium. Characterization of trophic factors for ORNs requires cell culture systems that are simple and easy to manipulate. We have compared four different cell culture preparations, using two different enzymes and two different media to develop a simple culture system of olfactory epithelial cells. Our preferred preparation, which produces partially purified olfactory epithelial cultures, uses trypsin dissociation and a serum-free keratinocyte growth medium (KGM) supplemented with insulin. These conditions support ORN survival up to 1 week. They also supported other elements of the olfactory epithelium such as Bowman's gland cells and horizontal basal cells. Olfactory epithelial cells predominate, while contaminating mesenchymal cells (glia and fibroblasts) are present in low numbers. Using these cultures, it was determined that insulin was required for ORN survival in vitro. The simplicity of the epithelial cultures will be useful for further studies of insulin and other ORN trophic factors.