Fragments of the 5'-flanking sequence of a human 2-5A synthetase gene were assayed for their ability to respond to interferon-alpha (IFN). Transient transfection assays in monkey cells demonstrated that the 5' boundary of the sequence required for IFN-regulated transcription is, at most, 155 nucleotides upstream from the presumed translational initiation codon. The 3' boundary of this sequence lies within a region of multiple transcription start sites preceded by no obvious TATA box. Binding assays, using a 40-bp probe derived from this IFN-responsive sequence, demonstrated the presence of three IFN-modulated, DNA-factor band shifts using nuclear extracts prepared from human and monkey cells. The induction of these complexes in human cells by IFN occurs with kinetics which closely parallel those previously observed for the transcriptional activation of the 2-5A synthetase gene by IFN. In vivo competition assays showed that the same 40-bp region which bound IFN-modulated factors could decrease the IFN-induced activity of a co-transfected 2-5A synthetase promoter; this fragment, regardless of its orientation, could confer IFN-inducibility on a heterologous promoter.