Alpha/beta interferons (IFN-alpha/beta) induce potent antiviral and antiproliferative responses and are used to treat a wide range of human diseases, including chronic hepatitis C virus (HCV) infection. However, for reasons that remain poorly understood, many HCV isolates are resistant to IFN therapy. To better understand the nature of the cellular IFN response, we examined the effects of IFN treatment on global gene expression by using several types of human cells, including HeLa cells, liver cell lines, and primary fetal hepatocytes. In response to IFN, 50 of the approximately 4,600 genes examined were consistently induced in each of these cell types and another 60 were induced in a cell type-specific manner. A search for IFN-stimulated response elements (ISREs) in genomic DNA located upstream of IFN-stimulated genes revealed both previously identified and novel putative ISREs. To determine whether HCV can alter IFN-regulated gene expression, we performed microarray analyses on IFN-treated HeLa cells expressing the HCV nonstructural 5A (NS5A) protein and on IFN-treated Huh7 cells containing an HCV subgenomic replicon. NS5A partially blocked the IFN-mediated induction of 14 IFN-stimulated genes, an effect that may play a role in HCV resistance to IFN. This block may occur through repression of ISRE-mediated transcription, since NS5A also inhibited the IFN-mediated induction of a reporter gene driven from an ISRE-containing promoter. In contrast, the HCV replicon had very little effect on IFN-regulated gene expression. These differences highlight the importance of comparing results from multiple model systems when investigating complex phenomena such as the cellular response to IFN and viral mechanisms of IFN resistance.