Previously, gonad-stimulating substance (GSS), which acts as a gonadotropin, was purified from radial nerves of the starfish Asterina pectinifera and its structure was elucidated. Here, the interaction of GSS with receptors was examined in ovarian follicle cells, a target of GSS. In competitive experiments using radioiodinated and radioinert GSS, highly specific binding was observed in the microsomal/plasma membrane fraction of follicle cells. GSS scarcely bound in the cytosolic fraction. Scatchard plots showed the numbers of binding sites (NBS) in whole homogenate and the crude membrane to be 1.65 and 3.42 pmoles/mg protein, respectively. Dissociation constant (K_d) values in these two preparations were almost the same at about 0.6–0.7 nM. Furthermore, it was shown that GSS stimulated adenylyl cyclase activity in follicle cell membranes in a dose-dependent manner that required GTP. Immunoblotting with specific antibodies for G-protein subunits after SDS-PAGE of the membrane preparation showed both stimulatory (Gs) and inhibitory (Gi) regulatory α-subunits for adenylyl cyclase and a β-subunit. The results strongly suggest that GSS interacts with G-protein-coupled receptors (GPCR) located in the follicle cell membrane to stimulate Gs-protein and adenylyl cyclase activity.