Vitellogenin (VTG) is a useful biomarker for detecting the estrogenic activity of chemicals in aquatic environments. However, little information is available on the regulatory mechanisms of the expression of each VTG subtype, particularly the relationship between expression patterns of VTG1/2 and estrogen receptor (ER) subtypes, such as ER α and ER β . In this paper, we measured VTG1 and VTG2 mRNA induction in male medaka liver, which was treated with ER α -selective ligand, (17 α , 20E)-3-hydroxy-17,20-[(1-methoxyethylidene)bis(oxy)]-19-norpregna-1,3,5(10),20-tetraene-21-carboxylic acid, methyl ester or ER β -selective ligand, 2-(4-hydroxyphenyl)-5-hydroxy-1,3-benzoxazole and investigated the characteristics of ER subtype function in VTG1 and VTG2 inductions. Hepatic VTG1 mRNA was induced by ER α -selective ligands at even low concentration and maximum increases were the same as for E2. VTG2 mRNA was also increased, but its levels were very low. On the other hand, ER β -selective ligands significantly increased VTG2 mRNA in the presence of ER α agonists. These results indicate that the expression of each VTG subtype is regulated by unique ER subtypes. VTG1 expression is only regulated by the action of ER α . In contrast, VTG2 expression is regulated by both ER α and ER β , with ER α being essential for VTG2 gene expression and ER β being essential for enhancement.