Estrogen plays a key role in the control of reproductive behavior and in the regulation of the neuroendocrine system. To elucidate the mechanisms by which it controls these functions it is important to understand how estrogenic effects are mediated. We have investigated the distribution of the two isoforms of the chicken estrogen receptor alpha (cER-alpha) protein; the previously characterized cER-alpha 66 and a new N-terminal truncated isoform, cER-alpha 61. Immunolocalization demonstrated the presence of cER-alpha 66 protein in hypothalamic areas, principally the nucleus septalis lateralis, bed nucleus striae terminalis medialis, nucleus preopticus medialis, and nucleus infundibuli hypothalami, and in the anterior pituitary gland. When the distribution of ER-alpha immunoreactive cells was compared using the antibodies H 222 (directed against the hormone-binding domain) and ER 221 (directed against the 21-amino acid N-terminus), no apparent differences could be detected. Because this immunocytochemical approach was not able to distinguish whether full-length cER-alpha 66 is the only isoform observed in the ER-positive regions or whether both cER-alpha receptor isoforms are present, SI nuclease assays were performed to compare the relative abundance in these regions of the two distinct classes of cER-alpha mRNA variants (A1-D and A2), which encode the cER-alpha 66 and cER-alpha 61 protein isoforms, respectively. In cockerels and hens, both variants of cER-alpha mRNA are expressed in the anterior pituitary gland and basal hypothalamus with a dominance of the mRNA that encodes cER-alpha 66, whereas the mRNA that encodes cER-alpha 61 was not detectable in the anterior hypothalamus. Therefore, because both receptor isoforms differ in their ability to modulate estrogen target gene expression in a promoter and cell type-specific manner, these differences may mediate the pleiotropic actions of estrogen in reproductive behavior and neuroendocrine functions.