Estrogens work by binding to and activating specific estrogen receptors (ERs). Although mammals have two major nuclear ER subtypes (ERalpha and ERbeta), three subtypes have been shown in teleost fish (ERalpha, ERbeta-I, and ERbeta-II). 17beta-Estradiol stimulates the production of an egg yolk precursor protein (vitellogenin) in the liver of oviparous species, including the goldfish. However, the functional involvement of the ER subtypes in this process is not fully understood. Here, using primary goldfish hepatocytes, we test the hypothesis that all three ER subtypes are functionally involved in the liver of goldfish by using RNA interference to specifically knock-down the different ER subtypes. The results suggest that ERalpha is induced by estradiol through activation of the ERbeta subtypes. This induction serves to sensitize the liver to further stimulation by estradiol. The knock-down results were supported by use of ER subtype specific antagonists. Sensitization by up-regulation of ERalpha is likely to be important for seasonal spawners such as goldfish, to bring about a change from somatic growth to reproductive development, and vitellogenesis. The novel data presented in this study provide strong support for the hypothesis that the goldfish ER subtypes play functional roles in the regulation of vitellogenin and ERalpha and provide a framework for the better understanding of ER signaling in fish and other vertebrates.