Members of the E2F transcription factor family are critical downstream targets of the tumor suppressor RB and are often deregulated and hyperactive in human tumors. E2F regulates a diverse array of cellular functions including cell proliferation and apoptosis. Recent studies indicate that E2F also regulates expression of upstream components of pivotal signal transduction pathways, thereby modulating the activity of these pathways. We show here that E2F modulates the activity of the JNK pathway via E2F-induced upregulation of JNK phosphorylation. Accordingly, downregulating E2F1and E2F3 inhibits sustained UV-induced JNK phosphorylation and ectopic expression of E2F1 or E2F3 induces JNK phosphorylation and activation. The mechanism by which E2F modulates JNK phosphorylation involves transcriptional induction of the kinase GCK, a MAP4K that can activate JNK indirectly. Hence, inhibition of GCK expression impairs E2F1-induced JNK phosphorylation. The JNK pathway is an important mediator of stress-induced apoptosis and we show here that inhibition of JNK expression or activity significantly hinders E2F1-induced apoptosis. Overall, our data identify the kinase GCK as a novel E2F-regulated gene and reveal a functional link between a central signaling pathway, namely the JNK pathway, and the transcription factor E2F.