MKN28-derived nonreceptor type of serine/threonine kinase/mixed lineage kinase 2 (MST/MLK2) directly phosphorylates and activates SEK1/MKK4/JNKK1/SKK1 in vitro, thereby acting as a mitogen-activated protein (MAP) kinase kinase kinase in the JNK/SAPK pathway (Hirai, S. -i., Katoh, M., Terada, M., Kyriakis, J. M., Zon, L. I., Rana, A., Avruch, J., and Ohno, S. (1997) J. Biol. Chem. 272, 15167-15173). The in vitro reconstitution system for the kinase cascade allowed us now to identify JNK/SAPK activators involved in the MST/MLK2-dependent activation of JNK/SAPK in vivo. We show that at least two distinct MST/MLK2-dependent JNK/SAPK activators are present in the fractionated COS-1 cell lysate, and that they appear to be SEK1/MKK4/JNKK1/SKK1 and MKK7/JNKK2/SKK4 by Western blot analysis. Notably, a majority of the MST/MLK2-dependent JNK/SAPK-activating activity is found in MKK7-containing fractions, whereas the MEKK1-dependent activity is comparably distributed in SEK1- and MKK7-containing fractions. Moreover, MST/MLK2 activates recombinant MKK7 more effectively than recombinant SEK1, whereas MEKK1 activates both to a similar extent. In addition, the deletion analysis on MST/MLK2 showed that the kinase domain is responsible for the determination of substrate specificity. These results provide a molecular aspect to the differential regulation of the two JNK activators by a variety of cellular stimuli.