GH receptor (GHR) mediates the anabolic and metabolic effects of GH. We previously characterized a monoclonal antibody (anti-GHR ext-mAb) that reacts with subdomain 2 of the rabbit GHR extra-cellular domain (ECD) and is a conformation-specific inhibitor of GH signaling in cells bearing rabbit or human GHR. Notably, this antibody has little effect on GH binding and also inhibits inducible metalloproteolysis of the GHR that occurs in the perimembranous ECD stem region. In the current study, we demonstrate that anti-GH Rext-mAb inhibits GH-dependent cellular proliferation and also inhibits hepatic GH signaling in vivo in mice that adenovirally express rabbit GHR, as assessed with our noninvasive bioluminescence hepatic signaling assay. A separate monoclonal antibody (anti-GHR mAb 18.24) is a sister clone of anti-GHR ext-mAb. Here, we demonstrate that anti-GHR mAb 18.24 also inhibits rabbit and human GHR signaling and inducible receptor proteolysis. Further, we use a random PCR-generated mutagenic expression system tomapthe three-dimensional epitopes in the rabbit GHR ECD for both anti-GHR ext-mAb and anti-GHR mAb 18.24. We find that each of the two antibodies has similar, but nonidentical, discontinuous epitopes that include regions of subdomain 2 encompassing the dimerization interface. These results have fundamental implications for understanding the role of the dimerization interface and subdomain 2 in GHR activation and regulated GHR metalloproteolysis and may inform development of therapeutics that target GHR. Copyright © 2011 by The Endocrine Society.