We describe the isolation and characterization of six immortal cell lines derived from primary cultures of olfactory epithelium (OE) purified from E15 mouse embryos. Cultured cells were immortalized using a replication-defective murine retrovirus containing the cDNA for human c-myc, in addition to a dominant selectable antibiotic resistance gene (neo). Cells that survived antibiotic selection and displayed process-bearing morphologies were expanded and analyzed for expression of molecular markers characteristic of olfactory receptor neurons, sustentacular cells, and olfactory ensheathing cells. Interestingly, all six cell lines expressed morphological and immunological properties of the ensheathing, or Schwann, cells of the olfactory nerve, but did not express markers characteristic of olfactory receptor neurons. Our results suggest that, while it is possible to generate immortalized OE cell lines using retrovirus-mediated oncogene transfer, there may be limitations to the types of cells that can be immortalized. In addition, we demonstrate the potential usefulness of immortalized OE cell lines for promoter-trap experiments to identify developmentally regulated genes. © 1993 Academic Press. All rights reserved.