Bacterial collagenase was injected into the ventricular cavity of the optic tectum of chick and quail embryos. Histological examination up to 6 days after enzyme injection revealed that the collagenase disrupted the pial basal lamina, which was evident by the fragmented distribution of basal lamina proteins at the pial surface of the midbrain and the brainstem. Although the disrupted basal lamina was not reestablished at later stages of development, the pial basal lamina of the newly developing neuroepithelium in the caudal part of the tectum was continuous and intact. Western blot analysis showed that the collagenase digested collagens but spared noncollagenous proteins. The disruption of the pial basal lamina caused the neuroepithelial cells to retract their pial end feet and caused tectal axons to exit the brain tissue into the adjacent mesenchyme. The vertical migration of neuroblasts to the pial layers of the tectum was inhibited, leading to a disruption of the tectal histogenesis. In the developing optic pathways, retinal axons were misguided at the optic chiasma and terminated in the head mesenchyme instead of the tectum. None of the abnormalities in histogenesis and axonal pathways were observed when the basal lamina was disrupted at a later stage of embryonic development. The present experiments demonstrate that the pial basal lamina has an important function during brain morphogenesis in restricting axons to the brain, providing an anchoring of the neuroepithelial cells to the pial surface, and allowing the formation of a defined cytoarchitecture of the brain.