A time course study on the appearance and distribution of cells immunoreactive for spot 35 protein, a novel cerebellar Purkinje cellspecific protein, was conducted in the developing cerebella of fetal and early postnatal rats by PAP immunohistochemistry. Spot 35-immunoreactive cells were first noted in the cerebellar anlage on the 17th embryonic day, appearing as large cellular aggregations in the mantle layer and a small number of elongated cells dispersed between the cell aggregations and the ependymal layer. As the development proceeded, the spot 35-immunoreactive cells gradually accumulated beneath the external granular layer. At birth, they were arranged compactly in 4-5 irregular rows to form a primitive Purkinje cell layer. During their subsequent development, immunostaining for spot 35 protein demonstrated the rearrangement of the Purkinje cells into a single row and the maturation of their somata, axons and dendrites. All these findings indicate that spot 35 protein is a specific marker for the cerebellar Purkinje cells, from their migrating stage throughout the course of their maturation. The present study further describes the transitory lamellar arrangement of spot 35-immunoreactive Purkinje cells located horizontally at a short distance away from the ependymal layer of the cerebellum on the 17th embryonic day. © 1986.