The mammalian olfactory neuroepithelium, lining part of the nasal cavity, retains into adulthood progenitor cells for the olfactory receptor neurons and other cell types in the epithelium. The details of cellular lineage relationships are not completely understood. In particular, the exact nature of the interactions between several progenitor cell types and their relationship to neurons is not known. Studies of this system have been hampered by the lack of cell culture models and insufficient cell-type-specific markers. Antibodies to the cytokeratins are fairly specific markers for one potential progenitor cell type, the dark basal cells of the olfactory epithelium. Keratin immunostaining was used to develop cell culture systems which contained large numbers of putative dark basal cells, using the soft nasal mucosal tissues of both newborn and adult rats. Media and substrate conditions were optimized. The conditions which supported growth of keratin-positive nasal cells for greater than one month, and allowed partial purification, suggested similarities between olfactory and skin keratinocytes. Immunostaining with a monoclonal antibody specific for sustentacular cells (SUS-1) showed a subset of these cells present in culture, with some cells double-labelled with anti-keratin. This staining confirms the olfactory origin of at least a subset of the cells, and supports the proposal that the majority of cells were the dark olfactory basal cells. This culture system gives novel insights into olfactory epithelial cell physiology, and allows culture of these cells for further studies examining regulation of differentiation.