Estrogen receptor-α (ER-α) and ER-β exhibit fine differences in their distributions in the rodent forebrain, and one such difference is observed in the paraventricular (PVN) and supraoptic (SON) nuclei. To investigate the functional significance of ER in these brain areas, we examined the neuropeptide characteristics of ER-expressing neurons in the PVN and SON of female rats by using dual-label immunocytochemistry. The distributions of ER-α immunoreactivity (ir) and ER-β ir were nonoverlapping in the PVN and SON. Nuclear ER-α ir was found in a population of thyrotropin-releasing hormone (TRH)-expressing neurons in the PVN (5.93% ± 1.20% SEM), but not in any other identified cell phenotype of the PVN and SON. The phenotype of neurons with the highest percentage expressing ER-β was found to be prolactin (PRL) immunoreactive in both the parvocellular (84.95% ± 4.11%) and the magnocellular (84.76% ± 3.40%) parts of the PVN as well as the SON (87.57% ± 4.64%). Similarly, most vasopressin-immunoreactive neurons were also ER-β positive in the PVN (66.14% ± 2.47%) and SON (72.42% ± 4.51%). In contrast, although a high percentage of oxytocin (OXY) neurons coexpressed ER-β in the PVN (84.39% ± 2.99%), there was very little ER-β/OXY colocalization in the SON. Low levels of corticotropin-releasing hormone neurons also expressed ER-β ir in the PVN (12.57% ± 1.99%), but there was no ER-β colocalization with TRH. In summary, these findings further support the possibility of direct effects of estrogen on neuropeptide expression and implicate estrogen involvement in the regulation of various aspects of neuroendocrine function. J. Comp. Neurol. 484:28–42, 2005. © 2005 Wiley-Liss, Inc.