To investigate in vitro organotypic brain slice triple culture of neocortex-striatum-substantia nigra of postnatal rats. Slices (300um) from 2-day-old Wistar rats were transferred into an incubator with insert of Millicell membrane and incubated in Hanks balanced solution. They were cultured for 0d, 10d, 20d and 30d and were observed under a converted microscope to evaluate the growth state. Dopamine neurons and axons were labeled by tyrosine hydroxylase (TH) immunofluorescence. The ability that the slices absorb EB was tested with LCM510. Axons of TH-positive neurons in the substantia nigra were grown into the striatum. On 20d, the striatum join together with the substantia nigra with no necrotic cells found. On 30d, 10 percent neurons died of EB and necrosis. It was demonstrated in our experiment that 20d triple cultures with normal pathway formed from the substantia nigra to the striatum and this provides a unique in vitro histological model for studying the mechanism of some neural retrograde diseases such as Parkinson disease.