The stress-responsive p38 and JNK MAPK pathways regulate cell cycle and apoptosis. A human MAPKKK, MTK1 (= MEKK4), mediates activation of both p38 and JNK in response to environmental stresses. Using a yeast two-hybrid method, three related proteins, GADD45α (= GADD45), GADD45β (= MyD118), and GADD45γ, were identified that bound to an N-terminal domain of MTK1. These proteins activated MTK1 kinase activity, both in vivo and in vitro. The GADD45-like genes are induced by environmental stresses, including MMS, UV, and γ irradiation. Expression of the GADD45-like genes induces p38/JNK activation and apoptosis, which can be partially suppressed by coexpression of a dominant inhibitory MTK1 mutant protein. We propose that the GADD45-like proteins mediate activation of the p38/JNK pathway, via MTK1/MEKK4, in response to environmental stresses.