Prothymosin alpha (ProTα) is an acidic nuclear protein implicated in several cellular functions including cell survival. ProTα is found in the central nervous system, but the regional and cell type-specific expression patterns are not known. In this study, our immunohistochemical analysis demonstrated that ProTα is expressed ubiquitously throughout adult brain with difference in the intensity of region-specific protein reactivity. Interestingly, the highest ProTα signals were observed in the brain regions relevant to neurogenesis, such as sub-ventricular zone, granular cell layer of dentate gyrus, as well as granule cell layer of olfactory bulb. Strong immunoreactivity was also found in habenula, ependymal cells lining the dorsal third and fourth ventricle, and in neurons in the Purkinje cell layer of cerebellum. We showed that ProTα was strictly localized in the nuclei of neurons, while it was found in the cytosolic space of astroglial and microglial processes and cell body in the adult brain. To clarify the phenomenon underlying cytosolic localization of ProTα in non-neuronal cells, ZVAD-fmk, a caspase-3 inhibitor, was delivered intracerebroventricularly in the brain. At the follow-up 24 h after ZVAD-fmk injection, we found that nuclear intensity of ProTα was significantly increased in astrocytes, whereas the ProTα expression was not affected in microglia. The present study would contribute toward better understanding of physiological and pathophysiological roles of ProTα in the brain.