This study was undertaken to investigate the developmental expression of osteopontin (OPN) in the rat brainstem and cerebellum by Northern blotting and in situ hybridization. The expression of OPN was noted in the mesencephalic Vth nucleus initially at embryonic day 16 (E16). At E20, the labeling extended into other brainstem nuclei including the cochlear, vestibular, facial motor, and hypoglossal nuclei. During the first week of postnatal life, the OPN signal in the brainstem increased markedly, and by P14, OPN expression was found in functionally diverse areas including motor-related areas, sensory relay nuclei, and the reticular formation. The adult labeling pattern was established in central neurons at this time. These results corresponded well with those from Northern blot analysis. On the basis of morphological and distribution criteria, the OPN signal in several nuclei appeared to be contained exclusively within neuronal soma. OPN expression in neurons occurred during the period of neuronal differentiation and increased with maturation. Our results therefore suggest that OPN contributes to developmental processes, including the differentiation and maturation of specific neuronal populations, in the rat brain.