The role of the matrix glycoprotein fibronectin in the formation of the external granular layer of the developing mouse cerebellum was investigated by in vitro studies of the binding of cerebellar cells to a fibronectin-coated culture substratum and by in vivo immunocytochemical localization of antiplasma fibronectin antiserum in cerebellar tissue. The adhesion of cells dissociated from embryonic and early postnatal mouse cerebellum is developmental stage-specific when the cells are plated on tissue culture substrata derivatized with human plasma fibronectin. Cells dissociated from mouse cerebellum at embryonic day 13 form cellular aggregates on insoluble plasma fibronectin. In contrast, cells dissociated from embryonic day 16 through postnatal day 7 cerebellum form a monolayer. Time-lapse video recordings reveal extensive cell movement of late embryonic and early postnatal cerebellar cells on insoluble plasma fibronectin. Late embryonic and early postnatal cerebellar cells bind to fibronectin but do not degrade the fibronectin substratum. Immunocytochemical studies of the binding of antiplasma fibronectin antisera to cryostat sections of intact embryonic and early postnatal cerebellar tissue reveal a brightly stained region of endogenous fibronectin along the route of granule cell migration from the lateral caudal part of the neuroepithelium lining the fourth ventricle up onto the external surface of the cerebellar anlage. When the formation of the external granular layer is completed, the intense region of fibronectin is no longer visible.