GH3 cells, which normally release PRL in response to stimulation by TRH, have been stably transfected with rat GnRH receptor complementary DNA (GGH3-1' cells). Unlike the parent line, GGH3-1' cells express GnRH receptor, which can be measured in a radioligand assay using a metabolically stable GnRH analog. The number of receptors (11,000 +/- 2,800 receptors/cell; n = 3) and Kd (4.1 +/- 1.0 x 10(-8) M; n = 3), determined using a radioiodinated GnRH agonist, as well as binding inhibition values for GnRH agonists and antagonists and for unrelated substances suggest that this receptor is similar to those expressed in cell cultures derived from rat pituitaries, although the binding affinity is about 1 log lower in the former. Unlike GnRH-stimulated release of gonadotropins from primary pituitary cultures, which does not require protein synthesis and is not coupled to cAMP production, GnRH-stimulated PRL release from the transfected cell line is absolutely dependent on protein synthesis, and cAMP fulfills the requirements of a second messenger. The receptor appears to be coupled to adenylate cyclase-mediated PRL release through a cholera toxin-sensitive G-protein. These studies provide functional evidence to support the view that the cloned receptor is the physiological receptor for the releasing hormone, and that this receptor can differentially couple to G-proteins depending on their availability and accessibility in the target cell.