Mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) kinases (MEKKs) are serine/threonine kinases that are upstream regulators of MAPKs. Here, the role of the amino-terminal (N-terminal) domain of MEKK1-4 on the regulation of different intracellular signaling pathways, apoptosis, and cell proliferation has been assessed by comparing the responses induced by the full-length (FL) MEKKs to those induced by the kinase domains only. For each MEKK, the pattern of activation of NF kappa B, the ERK MAPK pathway, and the c-Jun N-terminal kinase (JNK) MAPK pathway markedly differed between the kinase domain and the FL form. Similarly, cell proliferation and apoptosis were differently regulated by the FL MEKK and the corresponding kinase domain. Our data show that the N-terminal domain of the MEKKs determines the specificity and the strength of activation of various intracellular signaling pathways and cellular responses.