Neuropeptide Y (NPY) is the most potent stimulant of feeding when administered by intracerebroventricular injection. Despite this, there is conflicting evidence as to its importance in the regulation of daily food intake and energy balance. It has been suggested that whilst it is important in the response to starvation it has little role in the regulation of daily food intake. To investigate the role of NPY in the regulation of food intake, anti-sense cRNA to NPY was expressed in the arcuate nucleus of adult male rats. The anti-sense NPY (AS-NPY) construct was initially tested in vitro and there was a decrease of approximately 50% in NPY release from anti-sense treated cells compared to controls (16.3±2.0fmol/L [AS-NPY] vs 37.3±7.7fmol/L [control], mean±SEM p<0.05). NPY release from hypothalamic explants from anti-sense injected animals was decreased by over 50% compared to those from controls at both 15 and 20 days after AAV injection (15 days 42%±6.5% [AS-NPY] vs 100%±36% [control], 20 days 41%±6% [AS-NPY] vs 100%±27% [control] mean±SEM, p<0.05). In a study lasting for 50 days, weight gain was significantly lower in anti-sense injected animals from day 16 (day 16: 6.25±1.10g [AS-NPY] vs 9.42±0.65g [control] mean±SEM, p<0.05) and remained so until the end of the study when they had gained approximately 40% less weight than controls (day 50: 52.0±9.6g [AS-NPY] vs 82.0±6.3g [control] mean±SEM, p<0.01). Cumulative food intake was significantly lower in the anti-sense injected animals from day 23 (day 23: 225.8±1.9g [AS-NPY] vs 250.6±8.7g [control], mean±SEM, p<0.05) and remained so until the end of the study (day 50: 834.5±14.8g [AS-NPY] vs 926.0±31.7g [control], mean±SEM, p<0.05). Similarly mean daily food intake was also reduced in the anti-sense injected animals (days 7–14: 24.9±0.4g/day [AS-NPY] vs 27.2±0.4g/day [control], mean±SEM, p<0.01). These data are supportive of a role for NPY in the regulation of daily food intake as well as in response to starvation.