We report here partial isolation and characterization of at least two GnRH-receptor binding factors from the ethanol:chloroform:acetic acid (ECA) extracts of rat testis. The displacement curve of defatted, steroid-free and desalted ECA extract was parallel to that of D-(leu)6-des(Gly)10-GnRH-EA in a GnRH-radioreceptor assay. Immunoaffinity chromatography on cyanogen bromide-activated Sepharose 4B beads covalently bound to an antibody raised against d-(lys)6-GnRH resulted in more than a hundredfold increase in receptor binding specific activity. Equivalent amounts of kidney extract after affinity chromatography showed no significant activity. Coincubation of the material purified by affinity chromatography with the labeled ligand did not result in significant peptidase degradation of the label, indicating that apparent displacement of the label in the receptor assay was not the result of cleavage of the ligand. HPLC of the material partially purified by affinity chromatography on a reverse phase 5 μm ODS column revealed two peaks of receptor binding activity. Preliminary estimates of molecular weights of these factors based on SDS-PAGE and gel filtration are 68,000 and 6,000 respectively, we conclude that there are at least two factors in rat testis with GnRH-receptor-binding properties that are chemically distinct from the native decapeptide.