α-Synuclein (αS), a presynaptic nerve terminal protein, is now known to be a major component of neuronal and glial cytoplasmic inclusions in α-synucleinopathies (Lewy body disease and multiple system atrophy). However, αS has not been identified in either neuronal or glial cytoplasm in formalin-fixed, paraffin-embedded tissue sections from the normal human brain. Previous studies have shown that pretreatment with either proteinase K or formic acid enhances αS immunoreactivity. The aim of the present study was, therefore, to study the effects of pretreatment with proteinase K and formic acid on αS immunoreactivity in vibratome sections of brain tissue taken from normal human subjects. In addition to presynaptic staining, αS immunostaining was recognized in neuronal perikarya in the pretreated sections; this immunoreactivity was more intense in sections taken from the deeper layers of the cerebral neocortex, the CA2/3 region of the hippocampus, and the substantia nigra. This pattern of αS expression coincides with the distribution of intraneuronal inclusions in αS transgenic animals as well as in human autopsy tissue taken from patients with Lewy body disease. Furthermore, intense immunoreactivity was also found in the cytoplasm of astrocytes and oligodendrocytes throughout the brain. These findings suggest that a significant amount of αS is also present in the neuronal and glial cytoplasm in the normal human brain.