Clonal cell lines of rat embryonic hippocampal origin have been developed by using retroviral transduction of temperature-sensitive simian virus 40 large tumor antigens. The cell lines undergo morphological differentiation at the nonpermissive temperature and in response to differentiating agents. Immunocytochemical analysis indicates that various lines are derived from progenitors of neuronal, glial, and bipotential lineages. Selected neuronal lines differentiate in response to diffusible factors released by primary glia, and one line of glial lineage supports the maturation of primary neurons in culture. Selected cell lines exhibit different patterns of neurotrophin gene expression that change after differentiation. In some lines, the relative levels of neurotrophin 3 and brain-derived neurotrophic factor message expression may reflect the developmental or regional differential expression seen for these genes in the hippocampus in situ. These hippocampal cell lines, which express markers indicative of commitment to neuronal or glial lineages, are valuable for studies of development and plasticity in these lineages, as well as for studies of the regulation of neural trophic interactions.