The relationships between binding affinity and the biological potency of eight GnRH agonist analogs were evaluated in isolated rat pituitary cells. For this purpose, binding affinity and biological activity were assayed under similar physiological conditions in medium 199, pH 7.4, and binding affinity was also measured under the standard conditions in hypotonic buffer at low temperature. Under physiological conditions, receptor binding affinity was consistently lower than when measured in the hypotonic Tris buffer usually employed for GnRH receptor studies. In the low temperature binding assay at 0 C, which provided a measure of the affinity constant without degradation, a difference of 20- to 30-fold was observed between native GnRH and its most potent analog, [D-Ser(t-Bu)6]des-Gly10-GnRH N-ethylamide. Modifications of the amino acid residues at both positions 6 and 10 of the decapeptide increased the binding affinity of GnRH analogs. When the receptor binding assay was performed at 37 C, the range of the apparent affinity constants was extended up to 60-fold. The affinity constants derived at 37 C were closely correlated with the biological potencies of the individual analogs measured in the same cell system. The effect of temperature on binding affinity was not significantly influenced by peptide metabolism, which was minor in the absence of horse serum from the incubation medium. At the pituitary level, the biological potency of the GnRH agonist analogs is predominantly determined by their higher receptor affinity, and reduced degradation is a less important aspect of the high biological activity of the superagonist analogs.