Binding of interferons (IFNs) to their cell surface receptors stimulates rapid translocation of cytoplasmic proteins to the nucleus and the expression of a variety of cellular genes within minutes. Translocated proteins subsequently bind to the interferon-stimulated response element (ISRE) located in the promoters of all IFN-activated cellular genes. We report here that ouabain, a specific inhibitor of the Na/K ATPase, selectively inhibited transcription of several IFN-α-induced cellular RNAs under conditions in which some other well-described signal transduction pathways remained intact. The latter included induction of human metallothionein 2A (HMT2A) by phorbol ester and induction of IP-10 RNA by IFN-γ. Ouabain itself induced RNA of the protooncogene c-fos which conversely was inhibited by IFN-α. Specificity of the ouabain effects on IFNα-induced RNAs with respect to a direct action on the Na/K ATPase was shown with a transfected monkey CV-1 cell line which expresses the ouabain-insensitive rat α1 subunit. Electrophoretic mobility shift assays (EMSAs) using nuclear extracts from ouabain-treated cells demonstrated that ouabain decreased IFNα-induced binding of the ISGF3 complex to the ISRE. Reconstitution experiments showed that this effect of ouabain is not due to the inhibition of IFNα activation of the ISGF3α subcomponent, which occurs in the cytoplasm, but a selective depletion of the ISGF3γ factor which in concert with activated ISGF3α induces interferon-stimulated gene (54 kDa) transcription. These findings imply that intracellular ion balance can selectively regulate the half-life of the ISGF3γ protein or the ability of this protein to complex with ISGF3α to activate IFNα-regulated cellular genes.