Although p38 MAPK is known to be activated in response to various environmental stresses and to have inhibitory roles in cell proliferation and tumor progression, its role in cell cycle progression in the absence of stress is unknown in most cell types. In the case of G(2)/M cell cycle control, p38 activation has been shown to trigger a rapid G(2)/M cell cycle checkpoint after DNA damage stress and a spindle checkpoint after microtubule disruption. In the course of our studies, we observed that p38 became actively phosphorylated, and its kinase activity increased transiently during G(2)/M cell cycle transition. Using an immunocytochemistry approach, the active form of p38 was found at the centrosome from late G(2) throughout mitosis, which suggests functional relevance for active p38 protein during mitotic entry. A closer examination reveals that p38 inhibition by pharmacologic inhibitors significantly accelerated the timing of mitotic entry. In addition, long term exposure of the inhibitor enhanced Cdc2 activity. These results indicate that p38 activity during G(2)/M may be involved in a mechanism for fine tuning the initiation of mitosis and perhaps transit of mitosis. Consistent with our previous findings, Cdc25B was phosphorylated on serine 309 at the centrosome during G(2)/M when p38 was active at this site; Cdc25B phosphorylation inhibits Cdc25B activity, and this phosphorylation was found to be p38-dependent. Taken together, our findings suggest that p38 regulates the timing of mitotic entry via modulation of Cdc25B activity under normal nonstress conditions.