AbstractStudies have demonstrated the involvement of mitogen‐activated protein kinase (MAPK) cascade pathways in the development of cerebral ischemic/hypoxic preconditioning (I/HPC). However, the role of mitogen‐ and stress‐activated protein kinase 1 (MSK1), an important downstream kinase of MAPK signaling pathways, in cerebral I/HPC is unclear. By using Western blot and immunostaining methods, we applied our unique “autohypoxia”‐induced I/HPC mouse model to investigate the effects of repetitive hypoxic exposure (H0–H6, n = 6 for each group) on phosphorylation and protein expression levels of MSK1 in the brain of mice. We found that the levels of phosphorylation on threonine 645 (Thr645) and serine 375 (Ser375) of MSK1, but not the protein expression, increased significantly both in hippocampus and in cortex of mice from H1–H6 groups (P < 0.05) over that of the normoxic group (H0, n = 6). Similarly, enhanced phosphorylations on Thr645 and Ser375 of MSK1 were also observed by immunostaining in both the cortex and the hippocampus of mice following three series of hypoxic exposures (H3). In addition, we found by using double‐immunofluorescence labeling that phosphorylated Thr645‐MSK1 colocalized with a neuron‐specific protein, neurogranin, in both cortex and hippocampus of I/HPC mice (H3). These results suggest that the increased neuron‐specific phosphorylation of MSK1 on Thr645 and Ser375, not protein expression, might be involved in the development of cerebral I/HPC in mice. © 2007 Wiley‐Liss, Inc.