To examine the role of the subfornical organ (SFO) in the osmotic activation of hypothalamic neurons, the responses of the SFO to osmotic stimulation were evaluated by using c-Fos protein immunohistochemistry. Numerous c-Fos-immunoreactive nuclei were found in the SFO of rats injected i.p. with hypertonic saline solution as early as 30 min after stimulation, and the effect lasted up to 3 h. Only a few c-Fos-positive cells were detected in the SFO of rats injected with isotonic saline. However, electrolytic lesions of the SFO did not prevent the osmotic activation of the hypothalamic paraventricular and supraoptic nuclei. These data suggest that the SFO and the hypothalamic magnocellular nuclei are simultaneously but separately activated by osmotic stress.