Ornithine decarboxylase was autoradiographically localized in the developing rat cerebellar cortex after intracisternal injection of [3H]alpha-difluoromethylornithine, a specific, irreversible inhibitor of the enzyme. At nine days of age, when cerebellar ornithine decarboxylase activity is maximal, autoradiographic grains were distributed over all layers of the cerebellar cortex and throughout the brain stem. Within cerebellar folia, the highest grain density was associated with the molecular layer, whereas the internal and external granule cell layers were less densely labeled. Enhancement of ornithine decarboxylase activity by intracisternally-administered isoproterenol correspondingly increased the autoradiographic grain density over each layer. Thus much of the polyamine biosynthetic capability needed to support neuronal and/or glial differentiation appears to be associated with the developing cell processes. The combination of [3H]alpha-difluoromethylornithine autoradiography with localized injection techniques provides a potentially powerful tool for the study of the involvement of polyamine biosynthesis in brain development.