Altered cellular levels and localizations of four distinct intracellular proteinases, cathepsins D, E, B, and L, with aging were studied in various rat brain tissues by enzymatic and immunohistochemical methods using discriminative antibodies specific for each enzyme. With regard to two aspartic proteinases, cathepsin E was barely detectable in all the brain tissues of young adult rats, including the cerebral cortex, the hippocampus, the neostriatum, and the cerebellum, whereas cathepsin D was ubiquitously found in these tissues. Two cysteine proteinases, cathepsins B and L, also existed in these tissues of young rats at the relatively high levels of activities. In aged rats, the cathepsin D levels in all of the brain tissues examined were about twice those of young rats. Cathepsin E was markedly increased in the cerebral cortex and neostriatum of aged rats, but not in the other tissues. The levels of cathepsin B were also increased significantly in the neostriatum of aged rats, but not significantly in the other tissues. In contrast, the activity levels of cathepsin L were strikingly decreased in all the brain tissues of aged rats. At the light microscopic level, the increased immunoreactivity of cathepsins D and E in the brain tissues of aged rats was eminent in both the neurons and the glial cells. By double-immunostaining technique, the cathepsin D-positive glial cells were mainly associated with reactive astrocytes, whereas the cathepsin E-positive glial cells were largely reactive microglial cells. Western blot analyses revealed that the molecular forms of cathepsins D and E increasingly expressed in the cerebral cortex of aged rats were similar to those of the respective normal mature enzymes. The increased immunoreactivity of cathepsin B in the neostriatum of aged rats was also found in both the neurons and the glial cells. Despite the marked decrease of the cathepsin L activity in various brain tissues of aged rats, the immunostaining for this enzyme was not significantly changed, indicating the occurrence of the catalytically inactive form of the enzyme in these tissues. These results suggest that the increased levels of cathepsins D, E, and B and the decrease in cathepsin L activity in brain regions of aged rats are related to both the neuronal degeneration and the reactivation of glial cells during the normal aging process of the brain.