Distinct but partially overlapping signaling pathways mediate the response to extracellular vs. intracellular sources of dsRNA, by toll-like receptor 3 (TLR3) and retinoic acid-inducible gene-I/melanoma differentiated gene 5 (RIG-I/mda-5), respectively. Different cell types signal through these pathways to widely varying de grees. We previously observed that exposure to extracellular dsRNA, delivered by its addition to the culture medium, could induce the interferon (IFN)-stimulated gene 56 (ISG56) in human HT1080 fibrosarcoma cells, but not the HT1080-derived cell line, U3A, which lacks functional Stat1. In this study, we further investigated the nature of the dsRNA signaling defect in U3A cells. We show that a defect affecting basal TLR3 mRNA expression prevents U3A cells from responding to extracellular dsRNA. This defect does not impair dsRNA signaling in response to viral infection or transfected dsRNA. Although U3A cells are deficient in Stat1, we found that Stat1 was not required for basal TLR3 expression because other cell lines lacking Stat1 expressed TLR3. Moreover, restoration of Stat1 expression failed to restore TLR3 mRNA expression in U3A cells. However, treatment of Stat1-restored U3A cells with either IFN-beta or IFN-gamma induced TLR3 expression and restored responsiveness to extracellular dsRNA. Our results demonstrate that Stat1 is critical for IFN-induced, not basal, responsiveness to extracellular dsRNA.