Inhibition of hypothalamic nitric oxide (NO) decreases energy intake, and changes in hypothalamic NO synthase (NOS) have been observed in genetically obese rodents, but it is not known if NO is involved in the development of diet-induced obesity (DIO). We therefore measured changes in hypothalamic neuronal NOS (nNOS) in DIO and investigated effects of peripheral and central inhibition of NOS in this model. Expression of nNOS in relation to changes in nutritional state was measured by immunohistochemistry, with radiochemical detection. The effect of chronic intraperitoneal (i.p.) administration of the NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 50 mg/kg/day) on energy intake, bodyweight and hypothalamic nitric oxide content was assessed in both chow-fed and DIO animals. Twenty-four hour energy intake after acute intracerebroventricular (i.c.v.) of L-NAME was also measured. Diet-induced obese animals had a statistically significant 32% reduction in the number of nNOS-immunolabelled cells in the ventromedial hypothalamus compared to chow-fed controls. Intraperitoneal administration of L-NAME decreased hypothalamic NO content in both chow-fed and DIO. Energy intake was reduced by 16% in DIO over 16 days, whereas energy intake was only reduced by 11% in chow-fed animals, although both were statistically significant. L-NAME significantly reduced body weight gain in DIO but not in chow-fed rats. L-NAME administered i.c.v. decreased 24 h energy intake to a greater extent in DIO rats, by 18%, compared with a 10% reduction in chow-fed rats. Ventromedial hypothalamic expression of nNOS is sensitive to changes in nutritional state. Despite having reduced nNOS, dietary obese rats were more sensitive to the effects of NOS inhibition than lean controls, suggesting a role for NO in the development of hyperphagia and obesity in rats fed a palatable diet.