The use of alternative promoters represents an important mechanism for the regulation of growth hormone receptor (GHR) gene expression. Two promoters have been isolated previously for the GHR gene: the P1 promoter that drives liver-specific expression, and the P2 promoter that drives ubiquitous expression. In the present study, we isolated a third GHR promoter termed P3. The P3 promoter was GC-rich and TATA-less. The P3 promoter was able to drive the expression of a luciferase reporter gene in cell lines Hep G2, PLC/PRF/5, and BHK-21. In vivo, the P3 promoter initiated transcription from two major sites in exon 1C of the GHR gene in many tissues. In the adult bovine liver, the P3-transcribed GHR mRNA represented only 10% of the total GHR mRNA pool. In non-hepatic tissues such as kidney, skeletal muscle, mammary gland, and uterus, P3-transcribed GHR mRNA represented 30-40% of the total GHR mRNA pool. Within the bovine GHR gene, the P3 promoter was located immediately downstream from the P2 promoter. In transfected cells, the P2 promoter served as an enhancer for the P3 promoter. Existence and co-regulation of two ubiquitous promoters may be a mechanism for achieving a high level of expression of the GHR gene in multiple tissues.