There are two estrogen receptor (ER) subtypes in fish, Esr1 and Esr2 (formerly ERα and ERβ), and in some species the Esr2 subtype has two forms, Esr2b (formerly ERβ1) and Esr2a (formerly ERβ2 or ERγ). There is little information, however, on the different characteristics and functional significance of the two receptor subtypes in fish, and this is especially relevant for understanding the disruption of ER signaling by chemicals with estrogenic activity. In this study, the full-length cDNAs for esr1 (3167 base pairs [bp]) and esr2b (2318 bp), and a partial-length (267 bp) cDNA for esr2a, were cloned and characterized in fathead minnow (fhm; Pimephales promelas), and their patterns of expression established during development and in adults. Real-time polymerase chain reaction revealed some clear distinctions in the ontogenic and tissue expression of fhm esr1, esr2b, and esr2a, suggesting different functions for each ER subtype. Fhm ERs were expressed in brain, pituitary, liver, gonad, intestine, and gill of male and female fish, esr2b and esr2a were also expressed in muscle. Fhm esr1 and esr2b were expressed predominantly in the liver, whereas fhm esr2a was expressed predominantly in intestine and was lowest expressed in liver. Responses of the different hepatic ERs in male fathead minnow exposed to 100 ng estradiol/L differed, with a significant induction (5-fold) of fhm esr1 but no effect on esr2b or esr2a expression, suggesting different mechanisms of regulation for the different ERs. The detailed characterization of ERs in fathead minnow provides the foundation for understanding the molecular basis of estrogenic disruption in fish.