We examined the effects of hyperosmotic stress on the gene expression and activity of neuronal nitric oxide synthase (nNOS) in the preoptic/hypothalamic neurosecretory system of the euryhaline tilapia Oreochromis mossambicus (Mozambique tilapia) by means of semiquantitative RT-PCR and NADPHd histochemistry. Expression of nos1 was rapidly and transiently up-regulated in the preoptic region and hypothalamus in response to a salinity change (70% seawater, SW). Expression levels increased 4 h after the salinity change and then returned to basal levels within 8 h of the hyperosmotic challenge. NADPHd histochemistry revealed that positive magnocellular and gigantocellular preoptic neurons increased in number 4 h after the salinity change, while the number of parvocellular preoptic neurons reactive for NADPHd showed no significant change. These results indicate that the nNOS gene expression and NOS activity are stimulated in the preoptic/ hypothalamic neurosecretory system in response to hyperosmotic stress and suggest that NO influences neuronal responses to short-term osmotic stimulation in euryhaline fish.