BACKGROUND & AIMS: The toll-like receptor 9 (TLR9) agonist IMO-2125 is currently evaluated in clinical trials for chronic hepatitis C therapy. The aim of this study was to investigate the in vivo mode of action of a closely related compound, referred to as immunomodulatory oligonucleotide (IMO). METHODS: We analyzed the Jak-STAT pathway activation and induction of interferon-stimulated genes in the liver of wild type, interferon-alpha/beta receptor-deficient and interferon-gamma-deficient mice, after administration of IMO. RESULTS: IMO induced a prolonged activation of the Jak-STAT pathway and upregulation of interferon-stimulated genes in the mouse liver. Contrary to the response observed after interferon-alpha injection, the signalling induced by IMO was not abrogated following repeated administration. At early time points after IMO injection, STAT1 phosphorylation and interferon-stimulated gene induction required a functional interferon-alpha/beta receptor, whereas at the later time points, the activation was type I interferon-independent. Microarray analysis revealed that IMO induced a broad transcriptional response in the mouse liver. This included upregulation of cytokine and chemokine genes responsible for recruitment of IFN-gamma producers, such as T cells and natural killer cells. Interferon-gamma-deficient mice showed a transient response to IMO, demonstrating the central role of interferon-gamma in sustained activation of Jak-STAT pathway by IMO. CONCLUSIONS: The bimodal kinetics of response to IMO in the mouse liver are driven by the sequential endogenous production of type I and II interferons. The lack of refractoriness to IMO, combined with the long-lasting induction of interferon-stimulated genes, reveals a favourable pharmacodynamics profile of this novel TLR9 agonist for the treatment of chronic viral hepatitis.