This study examined the effects of an injection of 17β-estradiol (E₂) on the expression of estrogen receptor (ER) α and β subtypes in the olive flounder (Paralichthys olivaceus). Time- and dose-related effects of E₂ on ERα and ERβ mRNA expression were determined by RT-PCR. In the liver of males, the two ER transcripts were up-regulated at 24 h. In the liver of females, ERα and ERβ were up-regulated at 36 h with the highest dose. After E₂ treatment, increases in ERα and ERβ expression were observed in the testis and ovary at 36 h; RT-PCR analysis showed that this increase was dose-dependent. After E₂ treatment, the brain tissue of males showed lower levels of ERα and ERβ compared with the untreated control group, whereas the brain tissue of females showed no significant difference compared with controls. The results confirm the hypothesis that ER regulation is tissue-specific and may be involved in E₂-mediated regulation of reproduction in the olive flounder.