Circulating estrogens influence the electrical and biosynthetic activity of the hypothalamic magnocellular neurons which synthesize vasopressin or oxytocin and regulate body fluid homeostasis and reproduction. As none of these magnocellular neurons express nuclear estrogen receptor in the rat, the present study has combined estrogen receptor immunocytochemistry with retrograde tracing techniques to examine whether the first-order neurons projecting to magnocellular neurons in the supraoptic nucleus may be receptive to estrogen. Green fluorescent latex microspheres (50 nl) were injected into the supraoptic nucleus of five ovariectomized rats. The largest numbers of retrogradely-labelled cells expressing estrogen receptor immunoreactivity were detected in the organum vasculosum of the lamina terminalis, anteroventral periventricular nucleus and medial preoptic nucleus where approximately 15% of all retrogradely-labelled cells were estrogen receptor-immunoreactive. Other prominent sites where double-labelled cells were detected were the median preoptic nucleus, subfornical organ, ventrolateral division of the hypothalamic ventromedial nucleus and the brainstem nucleus tractus solitarii. Triple labelling experiments in the caudal medulla revealed that the estrogen-receptive neurons of the nucleus tractus solitarii and ventrolateral medulla projecting to the supraoptic nucleus were not noradrenergic. These findings show that sub-populations of neurons projecting to the supraoptic nucleus express estrogen receptors. This provides immunocytochemical evidence that estrogen may regulate the activity of magnocellular oxytocin and vasopressin neurons in an indirect, trans-synaptic manner by influencing the activity of first-order neurons projecting to the supraoptic nucleus. The predominance of estrogen-receptive lamina terminalis and preoptic area inputs to the supraoptic nucleus suggests respective sites of estrogen action on magnocellular neurons in modulating fluid balance and reproductive function.