Innate cellular antiviral defenses are likely to influence the outcome of infections by many human viruses, including hepatitis B and C viruses, agents that frequently establish persistent infection leading to chronic hepatitis, cirrhosis, and liver cancer. However, little is known of the pathways by which hepatocytes, the cell type within which these hepatitis agents replicate, sense infection, and initiate protective responses. We show that cultured hepatoma cells, including Huh7 cells, do not activate the interferon (IFN)-beta promoter in response to extracellular poly(I-C). In contrast, the addition of poly(I-C) to culture media activates the IFN-beta promoter and results in robust expression of IFN-stimulated genes (ISG) in PH5CH8 cells, which are derived from non-neoplastic hepatocytes transformed with large T antigen. Small interfering RNA knockdown of TLR3 or its adaptor, Toll-interleukin-1 receptor domain-containing adaptor inducing IFN-beta (TRIF), blocked extracellular poly(I-C) signaling in PH5CH8 cells, whereas poly(I-C) responsiveness could be conferred on Huh7 hepatoma cells by ectopic expression of Toll-like receptor 3 (TLR3). In contrast to poly(I-C), both cell types signal the presence of Sendai virus infection through a TLR3-independent intracellular pathway requiring expression of retinoic acid-inducible gene I (RIG-I), a putative cellular RNA helicase. Silencing of RIG-I expression impaired only the response to Sendai virus and not extracellular poly(I-C). We conclude that hepatocytes contain two distinct antiviral signaling pathways leading to expression of type I IFNs, one dependent upon TLR3 and the other dependent on RIG-I, with little cross-talk between these pathways.