The human GH (GH) gene family is specifically expressed in somatotrophs of the anterior pituitary and placental syncytiotrophoblast. Two nuclease hypersensitive sites, HS III and HS V, are associated with a region of chromatin located 28 and 30 kilobases (kb) upstream of the pituitary GH gene transcription initiation site (+1) in both pituitary and placenta nuclei. A role for this region in pituitary GH gene expression has been reported, but the potential relevance to placental gene expression has not been determined. Deletion analysis of a 5.2 kilobase (kb) region (nucleotides -27,568/-32,746) containing HS III to V-related sequences localized significant enhancer activity to a 574 base pair (bp) HS III fragment (nucleotides -27,676/-28,249) in multiple transfected cell lines. Four nuclease-protected regions (FP1-4) were identified in the 574 bp fragment. FP2 and FP3 were detected with placenta cell nuclear protein, while FP1 and FP4 were observed with placental and non-placental cell nuclear extract. Disruption of FP1 had no effect on heterologous promoter activity in transfected pituitary and placental cells, while loss of FP2 and FP3 resulted in modest increases in placental cells, reflecting the presence of repressor activity associated with these regions in vitro. In contrast, disruption of the FP4 region by mutation or deletion significantly reduced enhancer activity. As a result, 30-fold enhancer activity was localized to a 41 bp region in transfected placental tumor cells. Binding of candidate proteins AP-2 (FP3) and Elk-1 (FP4) was confirmed using competition assays with specific oligonucleotides and antibodies. Moreover, these factors were associated with the hyperacetylated HS III region in human pituitary (AP-2 and Elk-1) and term placenta (AP-2) chromatin. These data implicate AP-2 and ETS-domain family members in the regulation of the GH/CS locus, and raise the possibility that different complexes form in the HS III region in placenta and pituitary cells.