To date, there are very few in vivo studies addressing the role of Bcl-2 phosphorylation. In a model of neonatal hypoxic-ischemic (HI) brain injury, we characterized the spatial and temporal phosphorylation of Bcl-2 at serine-24 (PS24-Bcl-2), using a site-specific antibody. Very few cells positive for PS24-Bcl-2 were found in control animals, but the number increased during reperfusion in all investigated brain areas in the ipsilateral hemisphere after HI, particularly in the border region between intact and damaged tissue. The highest numbers were encountered 24 h post-HI. Phosphorylation of Bcl-2 at serine-24 coincided with cytochrome c release after hypoxia-ischemia and preceded caspase-3 activation. Injured neurons displayed a predominantly nuclear, but also mitochondrial, localization of PS24-Bcl-2 immunoreactivity. In conclusion, phosphorylation of Bcl-2 at serine 24 was induced by hypoxia-ischemia, presumably resulting in loss of its anti-apoptotic function.