Stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK), belonging to the mitogen-activated protein kinase family, plays an important role in stress signaling. SAPK/JNK activation requires the phosphorylation of both Thr and Tyr residues in its Thr-Pro-Tyr motif, and SEK1 and MKK7 have been identified as the dual specificity kinases. In this study, we generated mkk7(-/-) mouse embryonic stem (ES) cells in addition to sek1(-/-) cells and compared the two kinases in terms of the activation and phosphorylation of JNK. Although SAPK/JNK activation by various stress signals was markedly impaired in both sek1(-/-) and mkk7(-/-) ES cells, there were striking differences in the dual phosphorylation profile. The severe impairment observed in mkk7(-/-) cells was accompanied by a loss of the Thr phosphorylation of JNK without marked reduction in its Tyr-phosphorylated level. On the other hand, Thr phosphorylation of JNK in sek1(-/-) cells was also attenuated in addition to a decreased level of its Tyr phosphorylation. Analysis in human embryonic kidney 293T cells transfected with a kinase-dead SEK1 or a Thr-Pro-Phe mutant of JNK1 revealed that SEK1-induced Tyr phosphorylation of JNK1 was followed by additional Thr phosphorylation by MKK7. Furthermore, SEK1 but not MKK7 was capable of binding to JNK1 in 293T cells. These results indicate that the Tyr and Thr residues of SAPK/JNK are sequentially phosphorylated by SEK1 and MKK7, respectively, in the stress-stimulated ES cells.