The migration of endothelial cells in response to various stimulating factors plays an essential role in angiogenesis. The p38 MAPK pathway has been implicated to play an important role in endothelial cell migration because inhibiting p38 MAPK activity down-regulates vascular endothelial growth factor (VEGF)-stimulated migration. Currently, the signaling components in the p38 MAPK activation pathway and especially the mechanisms responsible for p38 MAPK-regulated endothelial cell migration are not well understood. In the present study, we found that p38 MAPK activity is required for endothelial cell migration stimulated by both VEGF and nongrowth factor stimulants, sphingosine 1-phosphate and soluble vascular cell adhesion molecule. By using dominant negative forms of signaling components in the p38 MAPK pathway, we identified that a regulatory pathway consisting of MKK3-p38alpha/gamma-MAPK-activated protein kinase 2 participated in VEGF-stimulated migration. In further studies, we showed that a minimum of a 10-h treatment with SB203580 (specific p38 MAPK inhibitor) was needed to block VEGF-stimulated migration, suggesting an indirect role of p38 MAPK in this cellular event. Most interestingly, the occurrence of SB203580-induced migratory inhibition coincided with a reduction of urokinase plasminogen activator (uPA) expression. Furthermore, agents disrupting uPA and uPA receptor interaction abrogated VEGF-stimulated cell migration. These results suggest a possible association between cell migration and uPA expression. Indeed, VEGF-stimulated migration was not compromised by SB203580 in endothelial cells expressing the uPA transgene; however, VEGF-stimulated migration was inhibited by agents disrupting uPA-uPA receptor interaction. These results thus suggest that the p38 MAPK pathway participates in endothelial cell migration by regulating uPA expression.