Cells respond to viral infection through induction of discrete, innate immune response pathways that lead to induction of interferons (IFNs) and other proinflammatory cytokines, as well as the direct induction of some IFN-responsive genes that mediate specific antiviral or immunomodulatory responses. To assess the classes of genes induced directly upon treatment of cells with double-stranded RNA (dsRNA), a mimic of viral infection, we made use of a mutant human cell line defective in responsiveness to dsRNA and IFN. P2.1 mutant cells were generated from a Jak1-minus, HT1080 fibrosarcoma-derived cell line (U4C) after extensive mutagenesis with the intercalating agent ICR191. We now demonstrate that P2.1 cells are defective in basal and induced expression of toll-like receptor-3 (TLR-3), which may contribute to their dsRNA-unresponsive phenotype. After transfection with a wild-type TLR-3 gene, P2.1 cells were largely responsive to a dsRNA challenge, as assessed by activation of NF-kappaB and IFN regulatory factors (IRFs) and induction of IFN-beta and other genes. Untransfected and TLR-3-transfected P2.1 cells were assessed for global dsRNA responsiveness in oligonucleotide gene array studies alongside parental U4C and HT1080 cells. Several distinct patterns of gene induction in response to dsRNA challenge were identified, including genes expressed in a TLR-3-dependent manner, genes that required an intact IFN feedback for expression, and dsRNA-responsive genes that appeared not to require TLR-3 for induction. These data support the hypothesis that TLR-3 is an important determinant of cellular responses to external dsRNA and demonstrate distinctions in the repertoires of dsRNA-regulated genes induced when the IFN-feedback loop is present or absent in cells.