Intraperitoneal administration of hypertonic saline is a potent stimulus to the “stress” responsive hypophysiotrophic parvicellular neurons of the hypothalamic paraventricular nucleus (PVN), as well as to magnocellular neurons of the hypothalamo-neurohypophysial system. Precise identification of the pathways gating information about the “stress” of intraperitoneal hypertonic saline to the PVN has not been ascertained earlier. In this study we demonstrate that intraperitoneal injection of hypertonic saline induces increased expression of c-fos immunoreactivity within neurons of the medial parvicellular division of the PVN, within the circumventricular organs surrounding the anteroventral tip of the third ventricle, and within the magnocellular neurons of the PVN and supraoptic nucleus. Also, neurons involved in conveying visceral information to the PVN, including the parabrachial nucleus, the nucleus of the solitary tract and the ventrolateral medulla responded with increased c-fos expression to the “stress” paradigm. Combined c-fos immunocytochemistry and retrograde tracing experiments with cholera toxin B (ChB) was used to identify neurons projecting to the PVN activated by the applied “stressor.” Neither the mere intracerebral presence of ChB nor intraperitoneal administration of isotonic saline influenced the number of c-fos immunoreactive nuclei in the brain. Dual immunocytochemistry revealed that intraperitoneal administration of hypertonic saline induced expression of c-fos immunoreactive nuclei in approximately half of the retrogradely labeled neurons projecting to the PVN from the anteroventral tip of the third ventricle (AV3v), including the subfornical organ (SFO) and the organum vasculosum laminae terminalis (OVLT). In the brainstem, the “stressor” induced expression of c-fos-IR nuclei in almost all of the retrogradely labeled cells of the ventrolateral part of the medulla oblongata (A1 and C1), while only about 25% of the ChB-labeled cells of the caudal part of the nucleus of the solitary tract (A2) were concomitantly immunoreactive to c-fos. Within the parabrachial nucleus, only 20% of the ChB-labeled cells were also immunoreactive for c-fos. The present results provide evidence that information about the “stress” of intraperitoneal hypertonic saline is conveyed to both magnocellular neurons projecting to the neurohypophysis and hypophysiotrophic parvicellular neurons the PVN via afferent projections from a variety of neurons in the osmosensitive anteroventral tip of the third ventricle and visceromotor neurons of the parabrachial nucleus, the ventrolateral medulla and the nucleus of the solitary tract.