Increased expression of tumor necrosis factor alpha (TNFalpha) has been shown in adult stroke models. However, its expression and relationship with neuronal apoptosis in neonatal rats with transient middle cerebral artery occlusion (MCAO) have not been clearly elucidated. We studied the expression and distribution of TNFalpha and neuronal apoptosis in a postnatal Day 10 rat MCAO model using reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot, immunohistochemistry, fluorescence double-labeling, and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) analyses. We found TNFalpha mRNA expression increased at 2h and was maintained at high levels until 24h after reperfusion. TNFalpha protein expression was significantly increased from 4 to 8h (p < 0.01) lasting through 24h (p < 0.05) after reperfusion compared to the sham controls. TNFalpha immunoreactive cells were colocalized to neurons in both the core and the penumbra areas of the ischemic cortex. However, apoptotic cells were mainly distributed in the penumbra area and colocalized to neurons as well as to TNFalpha immunoreactive cells in the ischemic cortex. Our findings suggest that TNFalpha expression increases after neonatal stroke and is associated with neuronal apoptosis after transient focal cerebral ischemia.