Enterostatin, a pentapeptide cleaved from procolipase, suppresses fat intake after peripheral and central administration. Chronic treatment of rats with enterostatin decreases body weight and body fat. The effect was greater than could be accounted by the reduction in food intake alone. Hence, we have investigated the effect of enterostatin on energy metabolism. Male Sprague-Dawley rats adapted to a high fat diet were implanted with lateral cerebral ventricular or amygdala cannulas. The metabolic effects were determined by indirect calorimetry. After habituation to the test cages, fasted rats were injected with either saline vehicle or enterostatin given either intraperitoneal (ip) (100nmoles) or intracerebral ventricle (icv) (1nmole) or onto specific brain regions [amygdala (0.01nmole) or paraventricular nucleus (PVN) (0.1nmole)]. Respiratory quotient (RQ) and energy expenditure were monitored over 2 hours. Ip enterostatin reduced RQ (saline: 0.81 ± 0.02 vs enterostatin: 0.76 ± 0.01) and increased energy expenditure by 44%. Icv enterostatin increased the energy expenditure without any effects on RQ whereas PVN enterostatin increased metabolic rate while preventing the increase in RQ observed in the control animals. In contrast, neither RQ nor energy expenditure was altered after enterostatin was injected into the amgydala. Enterostatin activated AMPkinase in primary cultures of human myocytes in a dose and time dependent manner and increased the rate of fatty acid -oxidation. These findings suggest that enterostatin regulates energy expenditure and substrate partitioning through both peripheral and central effects.