Thymosin beta (Tbeta) isoforms play an important role in the organization of the cytoskeleton by sequestering G-actin during development of the mammalian brain. In this study, we examined changes in the expression of Tbeta4 and Tbeta15 after transient global ischemia. Tbeta15 mRNA increased gradually in the dentate gyrus (DG) of the hippocampal formation from 3 h after reperfusion and peaked 9 h later. Similarly, a significant increase in Tbeta4 mRNA level was observed in the DG 12 h after reperfusion. Tbeta4 and Tbeta15 proteins were found in different cell types in control brains; Tbeta15 was expressed in a subset of doublecortin (DCX)-positive cells in the DG, whereas Tbeta4-IR was observed in DG neurons and nearby microglial cells. After ischemia, Tbeta15-IR was found in DG neurons and Tbeta4-IR in the reactivated microglial cells. Interestingly, Tbeta15-IR accumulated in the nuclei of CA1 neurons, which are vulnerable to ischemic insults. These results suggest that Tbeta4 and Tbeta15 function in different cellular contexts during ischemia-induced responses.