The extent of agonist-induced down-regulation of the LH/CG receptor (LHR) in human kidney 293 cells transfected with the rat LHR (rLHR) is much lower than in two Leydig tumor cell lines (MA-10 and R2C) that express the rodent LHR endogenously. This difference can not be attributed to differences in the recycling of internalized receptors, or in the replenishment of new receptors at the cell surface. It can be correlated, however, with the half-life of internalization of the bound agonist, which is approximately 60 min in Leydig tumor cells and about 100 min in transfected 293 cells. To determine whether the rate of internalization of the bound agonist affects down-regulation, we compared these two parameters in 293 cells expressing four rLHR mutants that enhance internalization and three mutants that impair internalization. We show that all four mutations of the rLHR that enhanced internalization enhanced down-regulation, while only one of the three mutations that impaired internalization impaired down-regulation. In addition, cotransfections of 293 cells with the rLHR-wt and three constructs that enhanced internalization (G protein-coupled receptor kinase 2, beta-arrestin, and arrestin-3) increased down-regulation, while a related construct (visual arrestin) that had no effect on internalization also had no effect on down-regulation. We conclude that the rate of internalization of the agonist-LHR complex is the main determinant of the extent of down-regulation of the LHR.