There are two estrogen receptor (ER) subtypes in fish, ERα and ERβ, and increasing evidence that the ERβ subtype has more than one form. However, there is little information on the characteristics and functional significance of these ERs in adults and during development. Here, we report the cloning and characterization of three functional ER forms, zfERα, zfERβ1, and zfERβ2, in the zebrafish. The percentages of identity between these receptors suggest the existence of three distinct genes. Each cDNA encoded a protein that specifically bound estradiol with a dissociation constant ranging from 0.4 nM (zfERβ2) to 0.75 nM (zfERα and zfERβ1). In transiently transfected cells, all three forms were able to induce, in a dose-dependent manner, the expression of a reporter gene driven by a consensus estrogen responsive element; zfERβ2 was slightly more sensitive than zfERα and zfERβ1. Tissue distribution pattern, analyzed by reverse transcription polymerase chain reaction, showed that the three zfER mRNAs largely overlap and are predominantly expressed in brain, pituitary, liver, and gonads. In situ hybridization was performed to study in more detail the distribution of the three zfER mRNAs in the brain of adult females. The zfER mRNAs exhibit distinct but partially overlapping patterns of expression in two neuroendocrine regions, the preoptic area and the mediobasal hypothalamus. The characterization of these zfERs provides a new perspective for understanding the mechanisms underlying estradiol actions in a vertebrate species commonly used for developmental studies.