An interferon-stimulated response element (ISRE)/interferon regulatory element (IRE) spanning nucleotide coordinates 1091–1100 is present in the enhancer 1/X gene promoter region of the hepatitis B virus (HBV) genome. In the context of a minimal promoter element, the enhancer 1/X gene promoter ISRE/IRE was shown to be a functional regulatory site capable of mediating interferon α- (IFNα) and interferon-stimulated gene factor 3 (ISGF3)-specific transcriptional activation in transient transfection analysis. The enhancer 1/X gene promoter ISRE/IRE was also shown to mediate interferon regulatory factor (IRF) 1 and IRF7 activation of transcription from a minimal promoter construct. In contrast, IFNα and the IRFs had minimal effect on HBV transcription and replication in the context of the viral genome in cell culture.