DJ-1, the product of a causative gene of a familial form of Parkinson's disease (PD), undergoes preferential oxidation of cysteine residue at position 106 (Cys-106) under oxidative stress. Using specific antibodies against Cys-106-oxidized DJ-1, we examined oxidized DJ-1 immunoreactivity (oxDJ-1 IR) in wild type and DJ-1 knockout mouse brain sections as well as human brain sections from cases classified into different Lewy body (LB) stages of PD and PD with dementia. In mouse brain, the oxDJ-1 IR was particularly enriched in the olfactory bulb, cerebellum, cortex, and hippocampus. The oxDJ-1 IR was evident in the substantia nigra of the midbrain of mouse and human. In neuromelanin-containing neurons and nerve fibers of the substantia nigra of human brain section, oxDJ-1 IR was prominently observed, and the LBs also showed oxDJ-1 IR. OxDJ-1 was detected in the astrocytes of the striatum. In addition, neurons and glias in the red nucleus of the midbrain and the inferior olivary nucleus of the medulla oblongata, which is related to the regulation of movement, showed clear oxDJ-1 IR. The biochemical properties of oxDJ-1 in frozen brain samples were further investigated using oxDJ-1 specific antibodies. Consistent with immunostaining, oxDJ-1 band intensity in high oxDJ-1 IR group were significantly higher than that in the tissues from low oxDJ-1 IR group. Our observations suggest the relevance of DJ-1 oxidation to the homeostasis in multiple sites of brain, including neuromelanin-containing neurons of the substantia nigra.