The ability of a factor obtained from a 105,000 × g supernatant of rat testis to inhibit the hCG binding to gonadal receptors was studied. This factor was partially heat stable, not steroid in nature, and presented a molecular weight under 12,000. Binding studies indicated that the hCG binding inhibitor interfered with the formation of the hormone-receptor complex mainly by competing with hCG for the same binding sites. Consequently, it was demonstrated that the inhibitor prevented the interaction of hCG and its receptor in both testicular and ovarian tissues. Under in vitro binding conditions, it was observed that as the quantities of the inhibitor increased during the first incubation with testicular homogenate, and after removing the inhibitor and washing the pellet, less and less of the added labeled hCG was bound during the second incubation step. On the other hand, the hormone already bound to the tissue during the first incubation could not be displaced by increasing amounts of the inhibitor in the second incubation. Thus, binding of the inhibitor, or hormone, to the tissue in the first step was not readily exchangeable with the subsequent inhibitor, or hormone, added in the second step. These findings were consistent with the view that binding to LH/hCG receptors may proceed in an irreversible manner.