c-Jun N-terminal kinase (JNK) is emerging as an important regulator of cell migration. Perturbing the JNK signaling pathway with three structurally and mechanistically distinct inhibitors that selectively target either JNKs themselves or the upstream mixed-lineage kinases, we found dramatic inhibition of membrane protrusion and cell sheet migration during wound closure in Madin-Darby canine kidney (MDCK) epithelial cell monolayers. Extension of lamellipodia is blocked from the earliest times after wounding in the presence of JNK pathway inhibitors, whereas assembly of non-protrusive actin bundles at the wound margin is unaffected. Inhibitors of the other mitogen-activated protein kinase (MAPK) pathways, the extracellular signal-regulated kinase and p38 MAPK pathways, only have comparatively weak or marginal inhibitory effects on wound closure. Multiple splice variants of both JNK1 and JNK2 are expressed in MDCK cells, and JNK1 and JNK2 are rapidly and transiently activated upon wounding. Phosphorylation of c-Jun does not appear relevant to MDCK wound closure, and membrane protrusion directly after wounding is not affected by inhibitors of RNA or protein synthesis. While most known substrates of JNK are transcription factors or proteins regulating apoptosis, our data indicate that JNK regulates protrusion and migration in a gene expression-independent manner and suggest an important cytoplasmic role for JNK in the control of cell motility.