Full-scale transcriptional activation of the mouse Gbp genes by gamma interferon (IFN-gamma) requires protein synthesis in embryonic fibroblasts. Although the Gbp-1 and Gbp-2 promoters contain binding sites for transcription factors Stat1 and IFN regulatory factor 1 (IRF-1), deletion analysis revealed that the Stat1 binding site is dispensable for IFN-gamma inducibility of Gbp promoter constructs in transfected fibroblasts. However, activation of the mouse Gbp promoter by IFN-gamma requires transcription factor IRF-1. Transient overexpression of IRF-1 cDNA in mouse fibroblasts resulted in high-level expression of Gbp promoter constructs. Unlike wild-type cells, IRF-1% embryonic stem cells lacking functional transcription factor IRF-1 contained very low levels of Gbp transcripts that were not increased in response to differentiation or treatment with IFN-gamma. Treatment of IRF-1% mice with IFN-gamma resulted in barely detectable levels of Gbp RNA in spleens, lungs, and livers, whereas such treatment induced high levels of Gbp RNA in the organs of wild-type mice. These observations suggest two alternative pathways for transcriptional induction of genes in response to IFN-gamma: immediate response that results from activation of preformed Stat1 and delayed response that results from induced de novo synthesis of transcription factor IRF-1.