The subfornical organ (SFO), median preoptic nucleus (MnPO), and organum vasculosum lamina terminalis (OVLT) which are associated with the lamina terminalis are important in the control of body fluid balance. Neurons in these regions express estrogen receptor (ER)-, but whether the ER- neurons are activated by hypertonicity and whether hypertonicity regulates ER- expression is not known. Using fluorescent, double-label immunocytochemistry, we examined the expression of ER--immunoreactivity (-ir) and Fos-ir in control and water-deprived male rats. In control animals, numerous ER--positive neurons were expressed in the periphery of SFO, in both dorsal and ventral MnPO, and in the dorsal cap of the OVLT. Fos-positive neurons were sparse in euhydrated rats, but were numerous in SFO, MnPO, and the dorsal cap of OVLT following 48 hrs water deprivation. Most ER--ir neurons in these areas were positive for Fos indicating a significant degree of co-localization. To examine the effect of dehydration on ER- expression, animals with and without lesions surrounding the anterior and ventral portion of the 3rd ventricle (AV3V) were water deprived for 48 hrs. Water deprivation resulted in a moderate increase in ER--ir in the SFO of sham-lesioned rats (p=0.03) and a dramatic elevation in AV3V-lesioned animals (p<0.05). This was probably induced by the significant increase in plasma osmolality in both dehydrated groups (p<0.001) rather than a decrease in blood volume, because hematocrit was significantly increased only in the sham-dehydrated animals. Thus, these studies implicate the osmosensitive regions of the lamina terminalis as possible targets for sex steroid effects on body fluid homeostasis.