We have examined the temporal changes and cellular localization of osteopontin (OPN) mRNA and protein in organotypic hippocampal slice cultures subjected to ischemia-like oxygen-glucose deprivation (OGD). The sequential induction pattern response consisted of neuronal and microglial OPN upregulation, followed by a later extended phase of expression in reactive astrocytes. OPN immunoreactivity after OGD matched the mRNA induction patterns. Activated microglia revealed OPN staining in focal deposits, whereas neurons and reactive astrocytes showed perinuclear staining with a punctate cytosolic pattern of OPN, typical of secreted proteins. These data demonstrated that the temporal and cellular patterns of OPN induction in reactive glial cells in this in vitro model closely correlated with that in the in vivo model, suggesting that OPN has a multifunctional role in the pathogenesis of ischemic injury.