Palytoxin is a novel skin tumor promoter that does not activate protein kinase C. Previous studies demonstrated that palytoxin stimulates a sodium- dependent signaling pathway that activates the c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK) in Swiss 3T3 fibroblasts. In this study we show that a JNK kinase known as the stress-activated protein kinase/extracellular signal-regulated kinase-1 (SEK1) plays an important role in the regulation of JNK by palytoxin. We found that palytoxin stimulates the sustained activation of both JNK and SEK1 in COS7 and HeLa cells. Transiently expressed SEK1 isolated from palytoxin-treated cells can phosphorylate and activate JNK, which, in turn, can phosphorylate c-Jun. Furthermore, expression of a dominant negative mutant of SEK1 blocks activation of JNK by palytoxin. Sodium appears to play an important role in the regulation of JNK and SEK1 by palytoxin. Activation of JNK and SEK1 by palytoxin, but not anisomycin, requires extracellular sodium. Complementary studies showed that the sodium ionophore gramicidin can mimic palytoxin by regulating JNK and SEK1 through a sodium-dependent mechanism. Collectively, these results demonstrate that palytoxin stimulates a sodium-dependent signaling pathway that activates the SEK1/JNK/c-Jun protein kinase cascade.