A subset of DNA sequences in the 5'-flanking DNA of the human growth hormone (hGH) gene was examined by protein-DNA binding and gene transfer-expression experiments. Two adjacent cis-acting elements (I and II) were identified between nucleotides -308/-235 of the hGH gene that modulated the expression of a linked reporter gene in transfected HeLa cells. Elements I and II repressed gene expression whereas element II alone activated it. HeLa whole cell extracts contain two factors that bind hGH DNA carrying elements I and II. Factor I binds to single-stranded DNA, and its binding is correlated with repression of gene expression. Factor II binds between nucleotides -275/-257 of the hGH gene. This region is homologous to the binding site for the adenovirus major late transcription factor, and factor II binding to hGH DNA is competed by adenovirus major late promoter DNA, indicating that the hGH and major late adenovirus promoters share a transcription regulatory element.