While considerable information is available on the physiological effects of estrogen, much less is known about the regulation of estrogen receptor (ER) subtypes, particularly in non-mammalian vertebrates. Using goldfish as primary experimental model, we investigated sex- and tissue-specific homologous regulation of ER subtypes (ERα, ERβI, and ERβII) by estradiol in vivo, in the liver and gonads. Treatment with estradiol, significantly upregulated transcript levels for all three types of ERs (ERα, ERβI, and ERβII) in the goldfish ovary and testis. In the goldfish liver, treatment with estradiol significantly increased ERα, ERβI transcript levels without affecting ERβII. In all cases increased ER transcript level was correlated with increased ER protein level determined by Western blot analysis, although we are not able to distinguish between ER subtypes. The results provide strong support for the hypothesis that homologous regulation of ERs is tissue- and gender-specific, and may be a mechanism for estrogen-mediated regulation of reproduction in goldfish. Mol. Reprod. Dev. 74: 1105–1112, 2007. © 2007 Wiley-Liss, Inc.