The GHRH receptor (GHRH-R) acts as a critical molecule for proliferation and differentiation of somatotrophic pituitary cells. A role in the pathogenesis of GH hypersecretion and GH deficiency has been implicated. We investigated structure and regulation of the human GHRH-R gene. A genomic clone including approximately 12 kb of 5'-flanking region was isolated. The gene is of complex structure consisting of more than 10 exons. Two kilobase pairs of the promoter were sequenced, and putative transcription factor binding sites were identified. The transcription start site was defined by ribonuclease protection assay. Transcriptional regulation was investigated by transient transfections using promoter fragments ranging in size from 108-1456 bp. GHRH-R promoter (1456 bp) directed high levels of luciferase expression in GH4 rat pituitary cells whereas no activity was detected in JEG3 chorion carcinoma cells or COS-7 monkey kidney cells. A minimal 202-bp promoter allowed pituitary-specific expression. Its activity in COS-7 cells is enhanced by cotransfection of the pituitary-specific transcription factor Pit-1. We did not find any regulation of the GHRH-R promoter by forskolin, phorbol-myristate-acetate, or T3. Glucocorticoids lead to a significant stimulation, and estrogen leads to a significant inhibition. Further mapping suggests a glucocorticoid-responsive element between -1456 and -1181 and an estrogen-responsive element between -202 and -108. These studies demonstrate the complex nature of the human GHRH-R gene and identify its 5'-flanking region. Furthermore, specific activity of the promoter and regulation by various hormones are demonstrated.