Infections of bacteria and viruses induce host defense reactions known as innate responses that include the production of cytokines and chemokines. The production of type I interferon (IFN) is known to be induced by viral double-stranded (ds) RNA or bacterial lipopolysaccharide (LPS). Although important functions for the transcription factors NF-kappaB and interferon regulatory factor-3 (IRF-3) are indicated, the molecular signals leading to the activation of IFN genes have yet to be elucidated. We provide several lines of evidence that LPS and dsRNA trigger distinct intracellular signals upstream. Notably, our investigation revealed a critical function for TIRAP/MAL, a signaling adapter for Toll-like receptor (TLR) 4, in LPS-induced but not dsRNA-induced activation of IRF-3. These results highlight cross-talk between TLR-mediated and virus/dsRNA-induced signals resulting in activation of the IFN system.