Viral infection or double-stranded (ds) RNA induce interferons (IFN) and other cytokines. Transcription factors mediating IFN induction are known, but the signaling pathways that regulate them are less clear. We now describe two such pathways. The first pathway leading to NF-κB depends on the dsRNA- responsive protein kinase (PKR), which in turn activates IκB kinase (IKK) through the IKKβ subunit. The second viral- and dsRNA-responsive pathway is PKR independent and involves Jun kinase (JNK) activation leading to stimulation of AP-1. Both IKKβ and JNK2 are essential for efficient induction of type I IFN and other cytokines in response to viral infection or dsRNA. This study establishes a general role for these kinases in activation of innate immune responses.