The binding of the steroidal pheromone 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-P) to membranes prepared from the goldfish olfactory epithelium was investigated using an in vitro radioreceptor assay. Maximum specific binding occurred after a 60-min incubation at 4°C. Binding increased linearly with increasing amounts of tissue. 17α,20β-P binding saturated at a ligand concentration of approximately 4.0 nM, matching well with data obtained from electrophysiological recordings of the in situ sensitivity of the olfactory epithelium to 17α,20β-P. Scatchard analysis yielded a single class (r = 0.95) of high-affinity (Kd = 1.02 ± 0.06 nM), low-capacity (Bmax = 1.38 ± 0.24 pmol/mg protein) receptors. Olfactory epithelial membranes were highly enriched in 17α,20β-P receptors; binding was 10 to 20 times higher than in membranes prepared from gut, liver or brain. The specificity of binding was not absolute. Progestins (progesterone, 17α,-hydroxyprogesterone) and androgens (androstenedione, testosterone) competed with 17α,20β-P for binding; however, these steroids were less potent at displacing radiolabeled 17α,20β-P than was 17α,20β-P itself. Estrogens and glucocorticoids did not displace 17α,20β-P in this preparation. These results are consistent with the respective abilities of these compounds to elicit electro-olfactogram activity, and confirm the presence of membrane-bound steroidal receptors in olfactory tissue.