The growth hormone receptor (GHR) 1A promoter is responsible for transcription of the liver-specific GHR mRNA variant 1A in several mammalian species. We previously found that the region between nucleotide -218 and nucleotide -151 (relative to the major transcription start site) of the bovine GHR 1A promoter contained a binding site between -196 and -178 for the liver-enriched transcription factor hepatocyte nuclear factor 4alpha (HNF-4alpha) and that the same region might also interact with additional transcription factors in the liver. Using the -218/-151 region as bait to screen a bovine liver cDNA library in a yeast one-hybrid analysis, we identified chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) and HNF-4gamma, as well as HNF-4alpha, as binding proteins to the -218/-151 region. These binding proteins were also identified in a second yeast one-hybrid analysis using the -196/-178 region as bait, suggesting that COUP-TFII, HNF-4gamma and HNF-4alpha all bind to the -196/-178 region. Electrophoretic mobility shift assays confirmed the ability of these three transcription factors in bovine liver nuclear extracts to interact with the -196/-178 region. These interactions also appeared to exist in vivo, as the GHR 1A promoter containing the -196/-178 region could be recovered by immunoprecipitation of the bovine liver chromatin with antibody against COUP-TFII, HNF-4gamma or HNF-4alpha. In co-transfection analyses, each of these three transcription factors could activate GHR 1A promoter and the activation was dependent on the -196/-178 region. These results together identify COUP-TFII, HNF-4gamma and HNF-4alpha as transcription factors regulating GHR 1A promoter activity through binding to a common DNA element.