DNA damage and environmental stress activate signaling and induce genes involved in cell cycle and cell death. Expression of the Gadd45 protein is induced following DNA damage and other stress. Gadd45 is believed to play a role in growth arrest and possibly in cell death. The JNK signaling pathway is also activated by some DNA-damaging agents. This activation leads to phosphorylation and activation of transcription factors, such as c-Jun/AP-1 and ATF2, which mediate immediate early gene induction. Recently Gadd45 was suggested to be involved in JNK activation. However, as this suggestion relied on in vitro experiments and ectopic overexpression of Gadd45 protein, we examined whether physiological levels of Gadd45 that are induced following exposure to DNA damaging agents and stress can lead to JNK induction. We found that JNK activation by UV irradiation and anisomycin treatment precedes the induction of gadd45 mRNA by these agents. Gadd45 protein induction by methyl methanesulfonate also lagged behind JNK activation. The use of protein synthesis inhibitors suggested that newly synthesized proteins, including the stress-induced Gadd45, make only a marginal contribution to JNK activation. We also found that stresses such as γ irradiation induce Gadd45 and do not activate JNK in mouse fibroblasts. Therefore, stress-induced JNK does not depend on Gadd45 induction.