The discovery that the lateral hypothalamic area (LHA) might be important in modulating drinking behavior and fluid balance has led to numerous studies aimed at identifying the key neurotransmitters/neuromodulators and pathways involved. While past studies have demonstrated the presence of neuronal nitric oxide synthase (nNOS) within the LHA, its role in the regulation of fluid homeostasis is not known. In light of this, and the mounting evidence suggesting a role for nitric oxide in osmotic regulation within the hypothalamus, this study sought to determine the effects of 24- and 72-hours of water deprivation on nNOS protein expression within the LHA of the rat with immunohistochemistry. In euhydrated control animals we observed nNOS-like immunoreactivity throughout all levels of the LHA. Following 24 hours of dehydration the number of nNOS-like immunopositive neurons was significantly increased in the rostral but not the caudal regions of LHA. Seventy-two hours of water deprivation lead to further increases in nNOS-like immunoreactivity at different levels of the LHA. Interestingly, however, we observed increased nNOS-like immunoreactivity in the caudal regions of the LHA that was not evident after 24 hours of water deprivation. Double-labeling immunofluorescence histochemistry revealed that the nNOS-like immunoreactive neurons were not colocalized with the orexin-A-containing neurons. These results suggest that an osmotic challenge leads to an upregulation of nNOS immunoreactivity within discrete areas of the LHA. This altered neurochemistry within the LHA further highlights the potential importance of nitric oxide and the LHA in central regulation of fluid homeostasis.