The mammalian homeobox genes are a family of genes that are expressed at high levels in the nervous system and encode sequence-specific DNA-binding proteins and transcriptional factors. To understand the expression and function of the homeobox genes in the nervous system, we have studied the expression of the rat homeobox genes in the RT4 cell lines, which mimic specific properties of the neuronal, glial, and their bipotential progenitor cell types. Northern blot analyses indicate that the rat homeobox R5 and Rlb genes are expressed in all cell types in the RT4 cell family. The sizes and levels of RNA transcripts are comparable to those observed in vivo in rat spinal cord and rat embryos. In contrast, another rat homeobox gene Rla, which is expressed in vivo and is closely linked to the R1b gene, does not express detectable RNA species in the RT4 cell family. Thus, the rat homeobox genes are differentially regulated in the RT4 cell family. In protein blotting analyses, anti-R5 peptide antisera react with a 60-kDa protein in cell extracts from all the RT4 cell lines. Immunofluorescence experiments demonstrate that the homeobox R5 gene product is present in all cell lines and that the protein is predominantly localized in the nucleus. This suggests that the rat homeodomain proteins may function as transcriptional regulators in the RT4 cells.