To understand the roles of p38 mitogen-activated protein kinase (p38 MAPK) isoforms in adult mouse brain, in vivo activities and detailed expression patterns of two p38 isoforms, p38alpha and p38beta, were examined by using biochemical and immunohistochemical analyses. The result indicated that the activity of both p38alpha and p38b MAPKs in normal adult mouse brain was remarkably high, and the nuclear pool of the p38 isoforms was primarily responsible for most of the constitutive p38 MAPK activity in brain. Both p38alpha and p38beta were highly expressed in brain areas including cerebral cortex, hippocampus, cerebellum, and few nuclei of the brainstem. At the subcellular level, p38alpha was distributed in dendrites and in cytoplasmic and nuclear regions of cell body of neurons, which is in contrast to p38beta, since p38beta was preferentially expressed in nucleus of neurons. These results suggest that the p38 pathway may play an important role, not only in inflammation and neuronal cell death as previously suggested, but also in normal physiology of adult mouse brain.