Expression of the nuclear domain-associated proteins Sp100, PML, and NDP52, is enhanced by interferons (IFNs) on the mRNA and protein level. Increase both of Sp100 and PML mRNA is due to enhanced transcription of the corresponding genes which occurs independently of cellular protein synthesis immediately upon IFN-beta addition. Here, we describe the molecular cloning and functional analysis of the Sp100 promoter. DNA sequence analysis revealed potential binding sites for several constitutive and IFN-inducible transcription factors. Consistent with the absence of a TATA box and an initiator element, several transcription initiation sites were found. Transient expression studies identified an imperfect IFN-stimulated response element within the first 100 nucleotides upstream of the major transcription start site. This element rendered a heterologous promoter IFN-beta-inducible and bound IFN-stimulated gene factor 2 strongly but IFN-stimulated gene factor 3 only weakly. An IFN-gamma activation site approximately 500 base pairs upstream of the IFN-stimulated response element was found to bind three IFN-alpha/beta activation factors upon IFN-beta induction and conferred both type I and type II IFN inducibility upon a heterologous promoter. These data demonstrate a novel arrangement of a nonoverlapping IFN-gamma activation site and an IFN-stimulated response element mediating type I IFN inducibility, previously not reported for other IFN-stimulable promoters.