The mechanism of the interferon‐alpha (IFN‐α)‐induced antiviral response during hepatitis C virus (HCV) therapy is not completely understood. In this study, we examined the transcriptional response to IFN‐α in uninfected chimpanzees after single doses of chimpanzee, human, or human‐pegylated IFN‐α. Liver and peripheral blood mononuclear cell (PBMC) samples were used for total genome microarray analysis. Most induced genes achieved maximal response within 4 hours, began to decline by 8 hours, and were at baseline levels by 24 hours post‐inoculation, a time when high levels of circulating pegylated IFN‐α were still present. The rapid downregulation of the IFN‐α response may be involved in the transition between the observed phase I and phase II viral kinetics during IFN‐α therapy in HCV‐infected patients. The response to all three forms of IFN‐α was similar; thus, the reasons for previous failures in antiviral treatment of chimpanzees with human IFN‐α were not due to species specificity of IFN‐α. The response to IFN‐α was partially tissue‐specific. A total of 1,778 genes were altered in expression by twofold or more by IFN‐α, with 538 and 950 being unique to the liver or PBMC, respectively. Analysis of the IFN‐α and IFN‐γ responses in primary chimpanzee and human hepatocytes were compared as well. IFN‐α and IFN‐γ induced partially overlapping sets of genes in hepatocytes. In conclusion, the response to IFN‐α is largely tissue‐specific, and the response is rapidly downregulated in vivo, which may have a significant influence on the kinetics of antiviral response. (HEPATOLOGY 2006;43:961–972.)