During development, axons of the mammalian cerebral cortex show a high degree of selectivity in their growth into specific regions of the central nervous system (CNS). A number of studies have shown that growing axons are guided by permissive or inhibitory membrane-bound molecules. Cryostat sections of the developing brain provide a useful assay to investigate possible membrane-bound guidance cues because such cues are retained in their normal in situ locations in specific regions of the CNS. Moreover, cryostat sections can also be subjected to various treatments that affect membrane-bound molecules. Therefore, to determine the ability of such cues to regulate the growth and guidance of cortical neurites into specific brain regions at different stages of development, we used an in vitro assay system in which explants from newborn hamster cortex were plated onto various regions of cryostat sections from developing and adult hamster brain. Neurite outgrowth from cortical explants onto the cryostat sections was visualized with a fluorescent vital dye. Results showed first that cortical neurites grew robustly on neonatal cryostat sections but only sparsely on sections from adult hamster. Second, cortical neurites grew preferentially on regions of the neonatal sections such as the cortex, basal ganglia, brainstem, thalamus, and colliculus, which are either pathways or targets for cortical axons in vivo. In contrast, cortical neurites avoided growing on the cerebellum and olfactory bulb, which are neither targets nor pathways for cortical neurites in vivo. Results also showed that cortical neurites extending onto cortical regions of neonatal sections preferred to grow along the radial axis of the cortex. Finally, heat treatment of the neonatal sections drastically reduced cortical neurite outgrowth. Taken together, these results suggest that the growth and guidance of cortical neurites is influenced by substrate-bound, developmentally regulated, heat-sensitive guidance cues preserved in the cryostat sections.