The gene encoding a 67-kDa cytoplasmic guanylate-binding protein (GBP) is transcriptionally induced in cells exposed to interferon of either type I (alpha interferon [IFN-alpha] or type II (IFN-gamma). The promoter of the GBP gene was cloned and found to contain an IFN-alpha-stimulated response element, which mediated the response of the GBP gene to IFN-alpha. On the basis of transfection experiments with recombinant plasmids, two different elements were delineated. Both were required to obtain the maximal response of the GBP gene to IFN-gamma: the IFN-alpha-stimulated response element and an overlapping element termed the IFN-gamma activation site. Different proteins that act on each element were investigated, and their possible involvement in IFN-gamma-induced transcriptional regulation is discussed.