Pegylated interferon- (pegIFN-) has replaced un-modified recombinant IFN- for the treatment of chronic viral hepatitis because of its superior anti-viral efficacy that is generally attributed to improved pharmacokinetic properties. However, the pharmacodynamic effects of pegIFN- in the liver have not been studied. We analyzed pegIFN- induced signaling and gene regulation in paired liver biopsies obtained before treatment and during the first week after injection of pegIFN- in 18 patients. Despite sustained high serum concentrations of pegIFN- over the entire one-week dosing interval, IFN- signaling through the Jak-STAT pathway occurs only during the first day. PegIFN- induces hundreds of genes that can be classified into 4 clusters based on different temporal expression profiles. In all clusters, gene transcription is mainly driven by IFN stimulated gene factor 3 (ISGF3). IFN induced secondary transcription factors do not cause additional waves of gene expression. We could not confirm a role of un-phosphorylated STAT1 in prolonging IFN- induced gene transcription. Collectively, our results reveal that the major effects of pegIFN- in the liver are caused by an early and transient activation of ISGF3. Prolonging the serum half-life of IFN- does not necessarily improve its pharmacodynamic properties.