Within the cerebral and cerebellar cortices, neurons are organized in layers that segregate neurons with distinctive morphologies and axonal connections, and areas or regions that correspond to distinct functional domains. To explore the molecular underpinnings of pattern formation in layered regions of the CNS, we have characterized the patterns of expression of two homeodomain genes, Otx1 and Otx2, by in situ hybridization during embryonic and postnatal development in the rat. Otx1 and Otx2 are vertebrate homologs of the Drosophila gap gene orthodenticle, and are expressed during the development of the murine CNS (Simeone et al., 1992). Here we report that Otx1 mRNA is expressed in a subpopulation of neurons within cortical layers 5 and 6 during postnatal and adult life. This gene is also expressed by the precursors of deep-layer neurons within the developing cerebral ventricular zone, but is apparently downregulated by the progenitors of upper-layer neurons; Otx1 is never expressed by the neurons of layers 1–4. The spatial and temporal patterns suggest that Otx1 may play a role in the specification or differentiation of neurons in the deep layers of the cerebral cortex. Within the cerebellum, mRNAs for Otx1 and Otx2 are found within the external granular layer (EGL), but in three spatially distinct domains. During postnatal development, Otx1 is expressed within anterior cerebellar lobules; Otx2 mRNA is localized posteriorly, and a region of overlap in mid-cerebellum defines a third domain in which both genes are expressed. The boundaries of Otx1 and Otx2 expression correspond to the major functional boundaries of the cerebellum, and define the vestibulocerebellum, spinocerebellum, and pontocerebellum, respectively. Spatially restricted patterns of hybridization are observed early in postnatal life, at times that correspond roughly to the invasion of spinal and pontine afferents into the cerebellum (Arsenio-Nunes and Sotelo, 1985; Mason, 1987). These results raise the possibility that Otx1 and Otx2 play a role in cerebellar regionalization during early development.