Gonadotropin-releasing hormone receptors (GnRHRs) are expressed in gonadotropes and several extra-pituitary sites. They are assumed to be cell surface proteins but the human (h) GnRHR lacks features favoring plasma membrane localization and receptor location varies with cell type. When expressed in mammary (MCF7) cells, cell surface hGnRHR binding was much lower than that of mouse and sheep GnRHRs (type I GnRHRs without C-terminal tails), Xenopus (X) and marmoset type II GnRHRs (type II GnRHRs with C-tails) or chimeric receptors (type I GnRHRs with added XGnRHR C-tails). hGnRHR binding was higher in alphaT4 (gonadotrope-derived) cells and was increased less by C-tail addition. Whole cell levels of tagged human, Xenopus and chimeric GnRHRs were comparable (Western blotting) and confocal microscopy revealed that the hGnRHR is primarily intracellular (distribution similar to the endoplasmic reticulum marker, calreticulin), whereas most XGnRHR is at the plasma membrane, and adding the C-tail increased cell surface hGnRHR levels. A membrane-permeant antagonist increased cell surface hGnRHR number (>4-fold, t1/2 = 4 h) and also increased hGnRHR signaling and hGnRHR-mediated inhibition of proliferation. A more rapid increase in hGnRHR binding occurred when the temperature was raised from 4 to 37 degrees C (>5-fold, t1/2 = 15 min) and this effect was prevented by mutation to prevent signaling. Thus, cell surface GnRHR expression depends on receptor and cell type and the hGnRHR is primarily an intracellular protein that traffics to the cell surface for signaling in MCF7 cells. Manipulations favoring such trafficking may facilitate selective targeting of extra-pituitary GnRHRs.