We have prepared primary cultures of purified neonatal rat olfactory neurons. Dissociated olfactory epithelial cells are maintained in modified Eagle's medium with D-valine, cytosine arabinoside, and NGF. NGF is required for neuronal survival. Immunohistochemical staining is positive for the neuronal markers vimentin, olfactory marker protein, and neuron-specific enolase, but negative for the glial markers, glial fibrillary acidic protein, and S-100 protein. Physiologic concentrations of odorants stimulate cAMP accumulation in the cells. Because of their morphology, biochemical composition, and responsiveness to odorants, these cells should enhance olfactory investigations.