The interferon-alpha(IFN-alpha)-regulated hamster ISG-54 K gene, which is activated in hamster CHO-12 cells at least 40-fold, was isolated and the promoter region was characterized in detail. Sequence analysis revealed the presence of two elements, closely related to the interferon-stimulated-response-element (ISRE) consensus sequence [AGTTTCNNTTTC(CT)]. The putative ISRE-I sequence (GGTTTCAATTTCT) is located at position -97 to -85; ISRE-II (AGTTTTACTTTCT), which differs at three positions from ISRE-I, is found directly upstream of ISRE-I at position -110 to -98. In a transient transfection assay in CHO-12 cells the wild-type hamster ISG-54K-promoter-chloramphenicol-acetyl-transferase (CAT) reporter construct showed a 40-80-fold induction, offering an excellent model to study the functional properties of the two ISRE. To find out whether both elements were functional in interferon regulation of the promoter, selected point mutations were introduced in the -110 to -85 region and in flanking sequences. The (mutated) ISG-54 K promoter was linked to the CAT reporter gene and transiently expressed in CHO cells in the absence and presence of murine (Mu)IFN-alpha 6. Transfections showed that both the -97 to -85 (ISRE-I) and the -110 to -98 (ISRE-II) segment were needed for optimal interferon induction of the ISG-54 K promoter. However, ISRE-I has an approximately sevenfold stronger activity compared to ISRE-II. Sequential substitution of the three ISRE-I bases, which differ in ISRE-II showed that the T at position -105 causes the lower activity of ISRE-II. Transfection of ISG-54 K promoter constructs, in which ISRE-I was replaced by ISRE-II, which generates a promoter with two ISRE-II segments, and vice versa (two ISRE-I), provided further evidence for a role of both elements in IFN-alpha induction. Importantly, all data obtained in transfection studies show that the two ISRE cooperate synergistically. The mechanism of synergism is most probably an indirect interaction between transcription factors binding to the ISRE, because an increase in the spacial arrangement of the two ISRE with a complete helical turn or half a turn did not result in a substantial decrease in promoter activity.