Abstract: Objective:To study the conditions of isolation,cultivation,and to research the characteristics of proliferation,differentiation of rat embryonic neural stem cells（NSCs） in vitro.Methods: Embryonic hippocampus and subependymal layer tissues from embryonic rat（15-17d） was isolated.After being cultured for several days,these cells formed neuro-spheres in serum-free medium with epidermal growth factor（EGF）,basic fibroblast growth factor（b-FGF）,B27 and N2 supplement.Then these neuro-spheres were cloned and transferred by limiting dilution.Differentiated cells were identified by immunocytochemical method with type specific antibodies.Results: We could obtain many NSCs from rat embryonic brain tissues in vitro;they could be cultured and differentiated into neurons,astrocytes and oligodendrocytes;these cells stored in liquid nitrogen had the ability,too.Conclusions: Embryonic brain is rich of NSCs which have the ability of self-renewal and proliferation,and they remain the abilities to differentiate into neurons and astrocytes and oligodendrocytes when long-term cultured in serum-free medium.