Genetically distinct estrogen receptor (ER) subtypes (ERα and ERβ) play a major role in mediating estrogen actions in vertebrates, but their unique and overlapping functions are not entirely clear. Although mammals have 1 gene of each subtype (ESR1 and ESR2), teleost fish have a single esr1 (ERα) and 2 esr2 (ERβa and ERβb) genes. To determine the in vivo role of different ER isoforms in regulating estrogen-inducible transcription targets, zebrafish (Danio rerio) embryos were microinjected with esr-specific morpholino (MO) oligonucleotides to disrupt splicing of the exon III/intron III junction in the DNA-binding domain. Each MO knocked down its respective normal transcript and increased production of variants with a retained intron III (esr1 MO) or a deleted or mis-spliced exon III (esr2a and esr2b MOs). Both esr1 and esr2b MOs blocked estradiol induction of vitellogenin and ERα mRNAs, predominant hepatic genes, but esr2b was the only MO that blocked induction of cytochrome P450 aromatase B mRNA, a predominant brain gene. Knockdown of ERβa with the esr2a MO had no effect on estrogen induction of the 3 mRNAs but, when coinjected with esr1 MO, attenuated the effect of ERα knockdown. Results indicate that ERα and ERβb, acting separately or cooperatively on specific gene targets, are positive transcriptional regulators of estrogen action, but the role of ERβa, if any, is unclear. We conclude that MO technology in zebrafish embryos is an advantageous approach for investigating the interplay of ER subtypes in a true physiological context.