Exposure of primary murine embryonic fibroblasts to tumor necrosis factor (TNF) causes biphasic activation of the c-Jun NH₂-terminal kinase (JNK) signaling pathway. The early phase (30 min) of the response to TNF is a large and transient increase in JNK activity. This response is followed by a second and more sustained phase of JNK activation that lasts many hours. We employed a chemical genetic strategy to dissect the functional consequences of these two phases of JNK activation. We report that both the early and late phases of JNK activation contribute to TNF-induced gene expression. In contrast, the early transient phase of JNK activation (<1 hr) can signal cell survival, while the later and more sustained phase of JNK activation (1–6 hr) can mediate proapoptotic signaling. These data indicate that the time course of JNK signaling can influence the biological response to JNK activation.