Human (h) GH plays an essential role in growth and metabolism, and its effectiveness is modulated by the availability of its specific receptor [hGH receptor (hGHR)] on target cells. The hGHR gene has a complex 5'-regulatory region containing multiple first exons. Seven are clustered within two small regions: V2,V3,V9 (module A) and V1,V4,V7,V8 (module B). Module A-derived mRNAs are ubiquitously expressed whereas those from module B are only found in postnatal liver, suggesting developmental- and liver-specific regulation of module B hGHR gene expression. To characterize the elements regulating module B activity, we studied a 1.8-kb promoter of the highest expressing exon in liver, V1. This promoter was repressed in transfection assays; however, either 5'- or 3'-deletions relieved this, suggesting the presence of multiple negative regulatory elements. Six putative hepatic nuclear factor 4 (HNF-4) response elements were identified. We determined that HNF-4alpha is developmentally regulated in the human liver: HNF-4alpha2 and HNF-4alpha8 are expressed in fetal hepatocytes but only HNF-4alpha2 is expressed in postnatal liver. Transient transfection assays demonstrated that HNF-4alpha2 and HNF-4alpha8 have a similar dual effect on V1 transcription: activation via site 1 in the proximal promoter and repression through site 6, approximately 1.7 kb upstream. EMSA/electrophoretic mobility supershift assays and chromatin immunoprecipitation analyses confirmed these two sites are bound by HNF-4alpha. Based on these data, we speculate there are multiple regions working together to repress the expression of V1 hGHR transcripts in tissues other than the normal postnatal liver, and that HNF-4alpha is a good candidate for regulating V1 hGHR expression in the human hepatocyte.