Interferon-stimulated gene factor-3 (ISGF-3) is a multiprotein (113, 91, 84, and 48 kDa) transcriptional factor which regulates the expression of a specific set of genes, the interferon (IFN)-stimulated genes. In the studies presented here, we investigated the induction of synthesis of proteins of the ISGF-3 complex by IFNs. We report that both IFN-alpha and IFN-gamma induce a 3- to 5-fold increased expression of p91, p84, and p113 and their phosphotyrosine contents in a dose- and time-dependent manner. The IFN-mediated induction in the levels of p91 correlated well with the increased expression of steady-state levels of p91 mRNA by IFNs. Increased levels of p91 and p84 became detectable after 6 and 4 h treatment with IFN-alpha and IFN-gamma, respectively, and reached a maximum 5.2-fold at 18 h by IFN-alpha and 4-fold at 15 h by IFN-gamma. The levels of p113 were induced up to 3-fold at 15 h by IFN-alpha or IFN-gamma. The induction of ISGF-3 proteins by IFNs was accompanied by an increase in the accumulation of p91, p84, and p113 in the nucleus. The observed induction of increased expression of ISGF-3 proteins does not require continuous presence of IFNs, as removal of IFNs after 6 h of minimal treatment still resulted in a significant increase (2- to 4-fold) in the levels of expression of p91, p84, and p113 over an additional period of 12 h in culture, and induced proteins remained phosphorylated on tyrosine. The IFN-mediated increase in the synthesis of ISGF-3 proteins was blocked by Actinomycin D. Extension of these investigations to other human and mouse responsive cells, Daudi, Hela, and NIH3T3, also demonstrated significant increase in the levels of p91, p84, and p113 by interferons.