We have investigated the serotoninergic innervation of the ferret cortex from the day of birth to adulthood with immunohistochemical techniques. Due to the premature birth of ferrets, this period spans the entire generation of cells located within the upper cortical layers and their subsequent migration to their final positions. Already at birth, serotoninergic fibers innervate the developing cortex. This innervation is most dense within the marginal zone, the subplate region, and the lower portion of the cortical plate. As long as cell migration continues, serotoninergic fibers enter the expanding portions of the cortex. Only the region just below the marginal zone where newly arriving cells are added to the cortical plate is not innervated by the ingrowing fibers. When the bulk of cell migration ceases, during the third postnatal week, this gap disappears and the fibers gradually form a continuous innervation from the pia to the ventricle. As the cortex matures, the serotoninergic fibers become successively confined to the upper layers, to generate the adult pattern. In the adult ferret cortex, the highest innervation density is found within layers 1, 2, and 3, with a much sparser innervation within the lower layers (Voigt and de Lima, J. Comp. Neurol. 314:403-414, 1991). The dense innervation in the deep cortical layers is only transient, virtually disappearing toward adulthood. These results suggest that serotoninergic axons innervate cortical layers as soon as newly arriving cells reach their final positions within the cortex. This early innervation lends support to the idea that serotonin may play a role during development of the cerebral cortex.