Cloning of GnRH receptors from several animal species has made it possible to investigate receptor function using site-directed mutagenesis. However, many mutant GnRH receptors exhibit decreased ligand binding, which makes analysis of their ligand binding characteristics technically difficult. To increase the affinity of binding to the GnRH receptor, a novel tracer ligand, 125I-[His5,D-Tyr6]GnRH, was designed and synthesized to allow radioiodination at position 6 rather than the usual position 5. In competition binding assays, total binding of 125I-[His5,D-Tyr6]GnRH was higher than binding of a conventional tracer ligand, 125I-[D-Ala6,N-MeLeu7,Pro9NHEt]GnRH. The bindable fractions and specific activities of both peptides were similar, and the receptor binding affinities of the unlabeled peptides were indistinguishable. However, comparison of the radiolabeled peptides in saturation binding assays showed that the affinity of the peptide, 125I-[His5,D-Tyr6]GnRH, (Kd, 0.19 nM), was approximately 2-fold higher than that of the conventional tracer. The increased binding of 125I-[His5,D-Tyr6] GnRH has allowed the development of a sensitive GnRH receptor binding assay for analysis of mutant GnRH receptors that exhibit decreased ligand binding.