MEK kinase 1 (MEKK1) is a 196-kDa mitogen-activated protein kinase (MAPK) kinase kinase that, in addition to regulating the c-Jun NH₂-terminal kinase (JNK) pathway, is involved in the control of cell motility. MEKK1^−/− mice are defective in eyelid closure, a TGFα-directed process involving the migration of epithelial cells. MEKK1 expression in epithelial cells stimulates lamellipodia formation, a process required for cell movement. In addition, mouse embryo fibroblasts derived from MEKK1^−/− mice are inhibited in their migration relative to MEKK1^+/+ fibroblasts. MEKK1 is required for JNK but not NF-κB activation in response to virus infection, microtubule disruption, and stimulation of embryonic stem cells with lysophosphatidic acid. MEKK1 is not required for TNFα or IL-1 regulation of JNK or NF-κB activation in macrophages or fibroblasts. Thus, MEKK1 senses microtubule integrity, contributes to the regulation of fibroblast and epithelial cell migration, and is required for activation of JNK but not NF-κB in response to selected stress stimuli.