We isolated cDNA clones of two estrogen receptors (ER1 and ER2) from testis of the Japanese common goby (Acanthogobius flavimanus). The cDNAs of ER1 contained 3,796 nucleotides, including an open reading frame encoding 564 amino acids (M_r: 61.9 kDa); the cDNA for ER2 was 3,274 nucleotides long, with an open reading frame of 567 amino acids (M_r: 63.5 kDa). The deduced aminoacid sequences of ER1 and ER2 each had a characteristic ER structure consisting of five domains (A/B, DNA-binding, D, ligand-binding, and F) and were homologous to ERα and ERβ of other vertebrates. We therefore named ER1 and ER2 as goby ERα (gERα) and goby ERβ (gERβ), respectively. The DNA- and ligand-binding domains in each gER showed high similarity to the corresponding domains of other vertebrates. Analysis of the distribution of gERα and gERβ mRNAs in tissue by reverse transcription–polymerase chain reaction (RT-PCR) analysis revealed that gERα was expressed at high levels in brain and testis, and gERβ was expressed most strongly in testis. In situ hybridization showed that the mRNA of each gER was expressed mainly in the Sertoli cells of goby testis.