Transient transcriptional activation of the c-fos gene following serum stimulation of susceptible cells requires a conserved DNA element located 300 bp 5’to the mRNA cap site. A DNA-binding gel electrophoresis assay was used to detect a protein (s) in HeLa cell nuclear extracts that specifically binds to the 5’activating element. The protein recognizes a region of dyad symmetry within the 5’activating element, defined by binding competition, dimethylsulphate(DMS) interference and DNAase I and DMS protection studies. A single 22 bp synthetic copy of the dyad symmetry element will both compete efficiently for protein binding and restore serum regulation to c-fos” genes that lack the 5’activating element.