Prox-1, a prospero-related homeobox gene, is known to be an important transcription factor during embryogenesis. However, very little is known about Prox-1 expression and functions in the adult nervous system. Here we have investigated the expression pattern of Prox-1 mRNA and protein during postnatal brain development and in adult rat and mouse brains using in situ hybridization (ISH), immunohistochemistry (IHC) and Western blotting. In the developing and adult brain, we found prominent, but restricted Prox-1 mRNA expression in the dentate gyrus of the hippocampus, in some thalamic nuclei, notably in the anterior thalamus, and in the cerebellar cortex. Other brain regions, such as the hypothalamus and nuclei belonging to the midbrain, revealed a moderate level of Prox-1 mRNA expression. In developing cerebral cortex, Prox-1 mRNA was seen only in the thin layer under the pial surface postnatally, and the signal almost disappeared by the 28th postnatal day (PD). Using IHC and ISH approaches, we demonstrated rather restricted, but intense Prox-1 labeling in adult brain of both rat and mouse species. During postnatal brain development Prox-1 proteins by IHC, were below the detection limit at PD 14, while Prox-1 mRNA remained at a high level. Western blotting demonstrated the existence of two different variants of Prox-1 protein, one of which was about 20 kDa larger than ordinary size. During the first PDs, the larger variant predominated. At PD 14, neither protein variant could be detected. From PD 16 onwards the smaller variant started to predominate and by PD 30 the larger size protein had almost disappeared. The prominent but limited distribution of Prox-1 in the brain suggests its potentially important role during postnatal brain development and in adult CNS, which remains to be ascertained in future studies.