microRNAs are short RNA molecules that are often expressed in specific tissues and regulate a variety of developmental processes. We used locked nucleic acid probes in in situ hybridisation reactions to study the distribution of microRNA-449 (miR449) during mouse embryonic development in order to obtain clues about its function/s. Between E9.75 and E11.5, miR449 was found to be expressed specifically in the developing roof plate of the fourth ventricle within the domain of roof plate marker, Lmx1a. From E12.5 onwards, this expression became restricted to the epithelial cell layer of the fourth ventricle choroid plexus. MiR449 also became detectable specifically in the choroid plexuses of the lateral and 3rd ventricles at E13.5 and E15.5, respectively. Northern blot analysis of adult brain also showed a selective and enriched expression in the choroid plexus tissue. Potential target genes regulated by miR449 were selected for experimental validation in luciferase-reporter assays and the transcription factor E2f5, which regulates CSF production, was verified as a miR449 target gene. Taken together, these findings suggest that miR449 has a specific role in the development and functioning of choroid plexuses.