In our previous studies, we tentatively identified 17,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P) as a maturation-inducing hormone (MIH) in yellowtail (Seriola quinqueradiata) through in vivo and in vitro experiments. In this study, we investigated the binding sites for radioactive 17,20 beta-P and characterized the receptor binding to the ovarian plasma membrane in yellowtail undergoing first stage of maturation (FSM). Equilibrium binding sites for 17,20 beta-P have been detected within 1h incubation and the binding dissociated completely within 50 min at 4 degrees C and was pH dependent (optimum pH 7.8). Scatchard analyses of specifically bound 17,20 beta-P showed the evidence of a single class of high affinity binding sites (K(D)=22.9 nM), with limited capacity (B(max)=2.1 pmol/g tissue) to the ovarian membrane of yellowtail undergoing FSM. Competition results revealed that ovarian membrane receptor was highly specific for 17,20 beta-P. There was no other steroid competed strongly with the binding sites of [3H]17,20 beta-P, except 17,20 beta-P itself. On the other hand, 17,20 beta-P did not bind to the membrane prepared from maturationally incompetent (MI) and ovulation (OV) stages of oocytes. As the time proceeded after the stimulation of HCG, binding activity increased significantly (0.389+/-0.036 pmol/g tissue) in the ovarian membrane of maturationally competent (MC) oocytes by 12h postinjection. The binding activity was further significant (0.868+/-0.032 pmol/g tissue) at FSM by 24h postinjection and reached its peak (0.920+/-0.115 pmol/g tissue) temporarily at second stage of maturation (SSM) by 36 h postinjection and then sharply declined to the prestimulation levels during OV stage by 48 h postinjection. In addition to our previous findings, the present results indicate that 17,20 beta-P is the MIH in yellowtail.