Objective To improve the method  about the primary culture of rat brain microvascular endothelial cells in vitro.  Methods The SD rats aged from 4 to 6 weeks were chosen as research object. After craniotomy, washing and cutting, sieving, density gradient concentration of BSA, digestion of type Ⅱ collagenase and collagenase dispersive enzyme twice, the primary culture was carried out. The target cells were indentified by morphological abservation and immunocytochemical staining of  facter Ⅷ.  Results Cultured for 12 to 24 hours，the cells in vitro migrated outward from the microvascular section. The cells appeared polygonal-shaped，and proliferated in a clustered monolayer. the cell growth density reached 70％-80％ of the bottle bottom after 3 days，and arranged like cobbles. The correlation antigen of Ⅷ factor was positive，they reached confluence with over purity 99％.  Conclusion The method  is available that can successfully separate and cultivate microvascular endothelial cells of rat brains in vitro.