Therapy of chronic hepatitis C with pegylated interferon α (pegIFN‐α) and ribavirin achieves sustained virological responses in approximately half of the patients. Nonresponse to treatment is associated with constitutively increased expression of IFN‐stimulated genes in the liver already before therapy. This activation of the endogenous IFN system could prevent cells from responding to therapeutically injected (peg)IFN‐α, because prolonged stimulation of cells with IFN‐α induces desensitization of the IFN signal transduction pathway. Whether all types of IFNs induce refractoriness in the liver is presently unknown. We therefore treated mice with multiple injections and different combinations of IFN‐α, IFN‐β, IFN‐γ, and IFN‐λ. Pretreatment of mice with IFN‐α, IFN‐β, and IFN‐λ induced a strong expression of the negative regulator ubiquitin‐specific peptidase 18 in the liver and gut. As a result, IFN‐α signaling was significantly reduced when mice where reinjected 16 hours after the first injection. Surprisingly, both IFN‐β and IFN‐λ could activate the Janus kinase–signal transducer and activator of transcription (STAT) pathway and the expression of IFN‐stimulated genes despite high levels of ubiquitin‐specific peptidase 18. IFN‐λ treatment of human liver biopsies ex vivo resulted in strong and maintained phosphorylation of STAT1, whereas IFN‐α–induced STAT1 activation was transient. Conclusion: Contrary to the action of IFN‐α, IFN‐β, and IFN‐λ signaling in the liver does not become refractory during repeated stimulation of the IFN signal transduction pathway. The sustained efficacy of IFN‐β and IFN‐λ could be an important advantage for the treatment patients who are nonresponders to pegIFN‐α, through a preactivated endogenous IFN system. (HEPATOLOGY 2011;)