Abstract: Objective: To culture and identify neural stem cells from embryonic rat’s midbrain. Methods: The single cell suspensions derived from E14.5 rat embryonic mesencephlon were prepared and plated into the serum-free medium containing DMEM/F12（1:1）,EGF,bFGF,and B27supplement.After the primary neurosphere were formed,the single cell suspension of primary neurosphere were diluted by limiting dilution method,and single cell clone sphere of neural stem cell were obtained with successive proliferation.The neurospheres were labled by using immucytochemistry detection of Brdu and Nestin.The distinctive marker for neuron（NeuN）,astrocyte（GFAP） and DA neuron（TH） were used to determine the phenotype of the differentiaed cells.Rsults:Single cell neuropheres consisting lots of cells were isolated from E14.5 rat embryonic mesencephlon after two weeks culturing.Large amounts of single cell clone spheres derived from one neural stem cell were obtained by successive passage.The neuropheres exhibited both Brdu and Nestin positive（90%）.The differentiated cells expressed GFAP,NeuN,and a small amount of differented cells expressed TH. Conclusion: The cells are multipotent and have the ability to undergo self-renew.So they are the stem cells of the central nervous system and they can turn to TH⁺ cells through differentation.