Two vitellogenins (Vgs) were detected in serum from estradiol-17beta (E(2))-injected Japanese common goby (Acanthogobius flavimanus). Vitellogenins with molecular masses of 530 kDa (Vg-530) and 320 kDa (Vg-320) were purified, and used to raise specific antisera in rabbits. Sandwich enzyme-linked immunosorbent assays (ELISAs) for Vg-530 and Vg-320 were developed using the antisera and the isolated Vgs. The sensitivity ranges of these ELISAs were 1.25-160 ng/ml for Vg-530 and 0.26-66 ng/ml for Vg-320, and very low cross-reactivity was found with the alternate Vg in each assay. Treatment of male gobys with E(2) by injection and immersion induced both Vgs in sera in a dose-dependent manner. The mean concentrations of the Vgs increased from 10 ng/L E(2) exposure for three weeks. Serum concentrations of the two Vgs in field-collected maturing females increased in accordance with increment of E(2) level and ovarian development, and the mean concentrations of Vg-530 were higher than those of Vg-320 in maturing female. These results indicate that the sandwich ELISAs for Vg-530 and Vg-320 developed in the present study is useful as an assay system for surveys of estrogenic activity in coastal areas of Japan.