Treatment of human breast tumor cells with interferon-gamma (IFN-gamma) elevates caspase-8 expression and sensitizes these cells to death receptor-mediated apoptosis through the increased processing and activation of apical procaspase-8. We have characterized the human caspase-8 gene promoter and studied the transcriptional regulation of caspase-8 gene expression in MCF-7 breast tumor cells treated with IFN-gamma. Our findings show that IFN-gamma induces the up-regulation of caspase-8 mRNA expression through a protein synthesis-dependent mechanism involving the action of the IFN-gamma-inducible transcription factor interferon regulatory factor-1 (IRF-1) and without altering mRNA stability. The human caspase-8 gene promoter lacks recognizable TATA and CAAT boxes but contains a consensus Sp1 binding site. We have identified two major IFN-gamma-inducible transcriptional start sites in these cells by S1 nuclease mapping, confirmed by primer extension analysis. Deletion analysis of the promoter defined an 82-bp minimal region responsible for IFN-gamma-inducible promoter activity. In this region, we have identified an IFN-stimulated response element that is important for both the basal and IFN-gamma-enhanced transcriptional activities. Electrophoretic mobility shift assay analysis demonstrated that IFN-gamma induces a complex between an oligonucleotide probe containing the ISRE motif and IRF-1 over a similar time scale to the induction of caspase-8 mRNA. Exogenously expressed IRF-1 in MCF-7 cells up-regulated the activity of a luciferase reporter plasmid containing an 82-bp region of the caspase-8 promoter. These data define a new pathway through which IFN-gamma might control the sensitivity of tumor cell to death receptor-mediated apoptosis.