The expression of tumor necrosis factor alpha (TNFalpha) increases and participates in several central nervous system (CNS) disorders. However, its expression after transient middle cerebral artery occlusion (tMCAO) in mice is not fully discussed yet. Therefore, we examined gene expression and protein localization of TNFalpha in brain using real-time polymerase chain reaction (PCR) and immunostaining after 1 h tMCAO in mice. After 1 h of ischemic conditions, we observed an increase in the expression of TNFalpha mRNA from basal level. While the expression decreased immediately to control level after reperfusion, it increased again significantly at 24 and 48 h after tMCAO. TNFalpha-like immunoreactivity (TNFalpha-LI) was slightly detected in fibrous structures of the neurons before ischemia. After ischemia, TNFalpha-LI spread widely to the soma of neurons and became more abundant in the nerve fibers, including axonal and dendritic processes. Moreover, TNFalpha-LI was also expressed in the oligodendrocytes and, occasionally, in microglia/macrophages, but not in astrocytes 24 h after tMCAO. These results suggest that TNFalpha shows biphasic expression that corresponds with ischemia and reperfusion, and might play a role in various cells to regulate CNS disorders such as neuronal and oligodendritic cell death after transient ischemia.