A radioligand assay employing tritiated gonadotropin-releasing hormone, [3H-Pro9]GnRH or [3H-pGlu1]GnRH is used to investigate the binding of GnRH, its agonists, and its antagonists to male rat anterior pituitary homogenates. The tritiated GnRH purified by high pressure liquid chromatography and stored in 10 mM HOAc is stable for binding for at least 14 weeks. It is found that there is at least one high affinity site with an observed Kd of congruent to 2 nM and another low affinity site whose Kd is congruent to 1 microM. Only approximately 25% of the total specific binding is to the low affinity site. At room temperature, the binding is reduced to 50% of that at 0 C, and at 37 C, there is no measurable binding. Bacitracin has no effect on the binding at any temperature. Maximum binding occurs between pH 7.5--8.5. Quantitative relative binding potencies of several agonists and antagonists are given. These potencies closely parallel their biological potencies, but all antagonists have higher absolute binding affinities when compared to their potencies to inhibit GnRH-mediated LH secretion in vitro.