Signal transduction via mitogen-activated protein kinase pathways plays a key role in a variety of cellular responses, including cell proliferation, differentiation, tumor promotion, and cell death, c-Jun N-terminal kinases (JNKs) are identified as members of the mitogen-activated protein kinase family and are known to phosphorylate and activate several transcription factors, including c-Jun, ATF, and Elk-1. However, the role of JNK activation in tumor promotion is not yet defined. Because previous studies have indicated that exposure of JB6 Cl 41 cells to either 12-O- tetradecanoylphorbol-13-acetate (TPA) or tumor necrosis factor-α (TNF-α) results in cell transformation, we investigated the role of JNKs in this biological process by using dominant negative JNK1 and the cell transformation model JB6 Cl 41 cells. Incubation of Cl 41 cells with TNF-α led to cell transformation and activation of JNKs. Introduction of the dominant negative mutant of JNK1 into JB6 Cl 41 cells specifically inhibited TNF-α-induced activation of JNKs, but not Erks and p38 kinases. Most importantly, expressing dominant negative mutant JNK1 inhibited TNF-α- induced cell transformation but not TPA-induced cell transformation, our results directly demonstrated for the first time that JNK activation is required for TNF-α- but not TPA-induced cell transformation.