High efficiency nuclear protein extracts were prepared from uninduced and recombinant interferon (IFN-alpha 2-primed Sendai virus-induced HeLa S3 cells and analyzed for DNA binding proteins specific for the human interferon-beta regulatory element (IRE). Analysis of protein-DNA interactions by a gel electrophoresis DNA binding assay resolved three complexes (designated A, B, and C) specific for the IRE probe (-79 to -36) in uninduced and induced cells. Competition studies using greater than a 100-fold molar excess of unlabeled DNA fragments representing different IFN-beta promoter sequences localized the DNA binding domain to the region -79 to -64 (P1 probe), a region previously shown to be essential for virus-inducible gene expression. Other adjacent IFN-beta segments including the negative regulatory element (-65 to -30) as well as the SV40 enhancer, c-fos serum-responsive element, and IFN-alpha 1 promoter fragment (-131 to -71) were unable to compete for the IRE binding protein. The 16-base pair enhancer core (P1 probe) specifically bound similar amounts of protein from uninduced and induced extracts; a synthetic tetrahexamer, AAGTGA, competed efficiently for the binding to the P1 region. Competition studies indicated that adjacent upstream IFN-beta DNA sequences -94 to -78 (P5) also contained a sequence motif capable of binding the P1 protein. In vitro transcription of the IFN-beta template in the HeLa nuclear extracts was partially inhibited when increasing amounts of competitor IRE fragment were added to the reaction. These results demonstrate that multiple DNA sequence motifs within the IFN-beta regulatory element interact to bind transcription regulatory proteins which are present in normal and virus-infected HeLa cells.