Four cell lines, stably transfected with rat GnRH receptor complementary DNA, have been prepared from the lactotropic GH3 cell line. All four lines (as well as the parent line and a line transfected with the vector DNA) show extensive rosettes of circular polyribosomes, characteristic of high protein synthetic activity, although secretory granules are virtually absent; the rough endoplasmic reticulum (rER) cisternae were short and straight. Instances were observed in which the ER reaches to the plasma membrane, suggesting a possible nongranular secretory route. All four lines (but not the parent or a control transfected line) expressed GnRH receptors that were down-regulated (1-5 h, depending on the cell line) after exposure to 10 nM GnRH; receptors then recovered (2-7 h). This pattern is reminiscent of the GnRH receptor in the primary gonadotrope cell cultures. All cell lines released PRL (4-96 h) in response to a GnRH agonist (D-tBuSer6-des-Gly10-Pro9-ethylamide-GnRH), an event that was inhibited by all three major classes of Ca+2 ion channel antagonists (methoxyverapamil, 1,4-dihydropyridines, and diltiazem); in contrast, GnRH-stimulated LH release from pituitary-derived primary cultures is only inhibited by methoxyverapamil. One line became refractory to GnRH analog stimulation after 24 h, although the other three released PRL vigorously up to the longest time point examined (96 h). All four lines responded substantially more robustly to 1 microgram/ml Buserelin than to 1 microgram/ml TRH. All four lines produced inositol phosphate metabolites and released immunoassayable cAMP (24 h) in response to treatment with Buserelin. These cell lines are good models for understanding the mechanisms by which the GnRH receptor is coupled to second messenger systems and for comparing these mechanisms with TRH-receptor coupling in the same cell.