The regulatory actions of estrogen on magnocellular oxytocin (OT) and vasopressin (VP) neurons of the paraventricular (PVN) and supraoptic (SON) nuclei are well documented. To date it is still debated whether the effect of estrogens is exerted directly or mediated by estrogen-sensitive interneurons. Previous immunocytochemical (ICC) and in situ hybridization (ISH) studies detected either low levels or absence of the classical estrogen receptor (ER-alpha) in the PVN and the SON of the rat. The present experiments using a combined ICC and ISH method were undertaken to examine the expression of the recently cloned beta form of ER (ER-beta) in OT- and VP-immunoreactive (IR) neuronal systems of the rat hypothalamus. The results demonstrate that the highest cellular levels of ER-beta messenger RNA (mRNA) in OT-IR neurons can be visualized in the caudal portion of the PVN and in an area ventro-medial to the central core of VP-IR cells. These neurons were previously shown to project caudally to the brain stem and the spinal cord to regulate autonomic functions. In addition, the whole rostro-caudal extent of the PVN and the SON contained OT-IR neurons that coexpressed variable levels of ER-beta mRNA. Similarly, the presence of ER-beta mRNA was seen in a large population of VP-IR paraventricular and supraoptic neurons. In the SON, somewhat stronger hybridization signal was detected in VP-IR neurons as compared with OT-IR neurons. Together, these findings provide strong support for the concept that the functions of OT- and VP-IR neurons in the PVN and the SON are regulated directly by estrogen and that the genomic effects of estrogens are mediated by ER-beta.