We have studied the relationship of retinal afferents, glial cell processes, and neuronal cytoarchitectonics in the lateral geniculate nucleus (LGN) of two species: tree shrews (Tupaia belangeri) and ferrets (Mustela putoris). Both species are relatively immature at birth, allowing the development of these features to be studied in the perinatal period. Retinal afferents, visualized by intraocular injection of a wheat germ agglutinin/horseradish peroxidase conjugate (WGA-HRP), are apparently the first elements of the developing LGN to exhibit a characteristic layered pattern in tree shrews and ferrets. Some radial glia still remain in the LGN of both species as the retinal afferents are in the process of segregating. Glial cell processes were visualized immunohistochemically with antibodies to glial fibrillary acidic protein (GFAP) or vimentin. In both the ferret and tree shrew, layering of glial cell processes is first seen as the overlap of retinal terminal fields diminishes. In the tree shrew LGN, these bands of dense glial cell staining are seen in apparent future cellular layers, whereas in the ferret, glial cell banding appears in interlaminar zones. If one or both eyes are removed at birth in tree shrews (before LGN cell layers are formed), the glial cell pattern seen 1 week later is in accord with the distribution of surviving nerve cells. The glial processes do not appear to invade regions left by degenerating retinal terminals or dying LGN cells. Several days after the appearance of layered glial cell processes (in the tree shrew) or at about the same time as glial layering (in the ferret), the first interlaminar spaces develop between neuronal cells, marking the beginning of cytoarchitectonic lamination, with its distinctive alternating cell-rich and cell-poor zones. Over the next several weeks, LGN neurons in both species continue to segregate into characteristic layers until the final, adult pattern of neuronal lamination is evident; as this process is completed, glial cell lamination disappears. These observations suggest that glial cells may be involved in establishing the neuronal layers that characterize the mature LGN of many species.