Interferon (IFN)-inducible human MxA protein mediates resistance against influenza and several other RNA viruses. The MxA gene is under the control of type I IFN and, in certain cell types, is also directly activated by viruses. Here we show that in human macrophages, MxA mRNA levels are upregulated by very low doses of IFN-alpha in a dose-dependent manner. A similar, albeit much weaker, dose-dependent induction was seen with IFN-gamma. The induction was rapid and independent of protein synthesis. Interleukin-6 (IL-6) or tumor necrosis factor-alpha (TNF-alpha) did not influence MxA mRNA levels alone or in combination with IFNs, in spite of the presence of putative response elements of these cytokines in the MxA promoter. We show that the promoter of the MxA gene contains two functional IFN-stimulated response elements (ISRE) near the transcription start site and one homologous ISRE-like element, which is apparently nonfunctional, further upstream. The two proximal ISRE sites are essential for IFN-alpha-induced transcription and appear to be binding sites for IFN-stimulated gene factor 3 complex. In addition, EMSA and DNAse I footprinting analysis demonstrated that Spl binds with high affinity to a region encompassing nucleotides -25 and -50 and, thus, may provide means of interaction with the basal transcriptional machinery.