Dopaminergic neurons in midbrain are subdivided into three subsets, forming mesostriatal, mesocortical and mesolimbic projections, respectively. The molecular mechanism specifying mesostriatal projection, which is important for understanding the pathogenesis of Parkinson's disease, remains unclear. To probe the role of axon guide molecule Ephrinb3 in inducing mesostriatal projection, we labeled mesostriatal and mesocortical subset DA neurons with fluorescent microspheres, and purified these subpopulation cells with fluorescence-activated cell sorting (FACS). Moreover, real-time PCR was performed to address the expression of Ephrinb3 in mesostriatal DA neurons, and fluorescence in situ hybridization (FISH) was used to further verify the expression of Ephrinb3 in labeled neurons. The results showed that mesostriatal DA neurons were successfully isolated with retrograde labeling and FACS. Real-time PCR showed that the expression of Ephrinb3 was higher in mesostriatal DA neurons than in mesocortical neurons. Also, Ephrinb3 could be detected in labeled neurons with FISH. Our results indicate that Ephrinb3 is directly involved in the specificity of mesostriatal projection. (C) 2010 Elsevier Inc. All rights reserved.