Stimulation of Toll-like receptors (TLRs) initiates potent innate immune responses through Toll–interleukin 1 receptor (TIR) domain–containing adaptors such as MyD88 and Trif. Analysis of Trif-deficient mice has shown that TLR3-dependent activation of the transcription factor NF-κB by the TLR3 ligand double-stranded RNA is Trif dependent. Here we investigated the 'downstream' signaling events that regulate TLR3-dependent Trif-induced NF-κB activation. Trif recruited the kinases receptor interacting protein (RIP)-1 and RIP3 through its RIP homotypic interaction motif. In the absence of RIP1, TLR3-mediated signals activating NF-κB, but not the kinase JNK or interferon-β, were abolished, suggesting that RIP1 mediates Trif-induced NF-κB activation. In contrast, the presence of RIP3 negatively regulated the Trif-RIP1–induced NF-κB pathway. Therefore, in contrast to other TLRs, which use interleukin 1 receptor-associated kinase (IRAK) proteins to activate NF-κB, TLR 3-induced NF-κB activation is dependent on RIP kinases.