The mechanism responsible for the induction of the 2-5A synthetase gene by Interferon-gamma (IFN-gamma) (type II) was studied in Friend leukemia cells. It was previously shown that activation of 2-5A synthetase gene expression by IFN-gamma in the 3Cl8 cell, a clone resistant to IFN-alpha,beta (type I), correlates with the formation of two major complexes, designated Fg and Fc, that bind to the interferon-stimulated responsive element of the gene. Conversely, in a clone resistant to both types of IFNs (3 gamma R8), no induction of DNA-protein complexes or of 2-5A synthetase gene expression was detected. In the present report the Fg complex has been characterized as including the interferon regulatory factor 1 (IRF-1), whereas the Fc factor, present also in control cells, has been characterized as composed of IRF-2. Incubation of cell extracts with antibodies to IRF-1 abolishes the formation of the Fg complex, and antibodies to IRF-2 abolish the formation of the Fc complex. Moreover, in the 3Cl8 cell, IFN-gamma is able to induce in few minutes the formation of a complex between a DNA element identified as the IFN-gamma activation site (GAS), present on the IRF-1 gene promoter, and the STAT1 protein. These findings suggest that in cells resistant to type I IFN, IFN-gamma is able, through the activation of the STAT1 protein, to induce the expression of the IRF-1 factor which in turn seems to be sufficient to transactivate the 2-5A synthetase gene.