Glucocorticoid (GC) treatment of rat or chicken embryonic pituitary (CEP) cells induces premature production of growth hormone (GH). GC induction of the GH gene requires ongoing protein synthesis, and the GH genes lack a canonical GC response element (GRE). To characterize cis-acting elements and identify trans-acting proteins involved in this process, we characterized the regulation of a luciferase reporter containing a fragment of the chicken GH gene (-1727/+48) in embryonic day 11 CEP cells. Corticosterone (Cort) increased luciferase activity and mRNA expression, and mRNA induction was blocked by protein synthesis inhibition. Through deletion analysis, we identified a GC-responsive region (GCRR) at -1045 to -954. The GCRR includes an ETS-1 binding site and a degenerate GRE (dGRE) half site. Nuclear proteins, including ETS-1, bound to a GCRR probe in electrophoretic mobility shift assays, and Cort regulated protein binding. Using chromatin immunoprecipitation, we found that ETS-1 and GC receptor (GR) were associated with the GCRR in CEP cells, and Cort increased GR recruitment to the GCRR. Mutation of the ETS-1 site or dGRE site in the -1045/+48 GH reporter abolished Cort responsiveness. We conclude that GC regulation of the GH gene during development requires cis-acting elements in the GCRR and involves ETS-1 and GR binding to these elements. Similar ETS-1 elements/dGREs are located in the 5'-flanking regions of GH genes in mammals, including rodents and humans. This is the first study to demonstrate involvement of ETS-1 in GC regulation of the GH gene during embryonic development in any species, enhancing our understanding of GH regulation in vertebrates.