Endocannabinoids are involved in the modulation of pain and hyperalgesia. In this study we investigated the role of the endocannabinoid system in the migraine model based on nitroglycerin-induced hyperalgesia in the rat. Male rats were injected with nitroglycerin (10 mg/kg, i.p.) or vehicle and sacrificed 4 h later. The medulla, the mesencephalon and the hypothalamus were dissected out and utilized for the evaluation of activity of fatty acid amide hydrolase (that degrades the endocannabinoid anandamide), monoacylglycerol lipase (that degrades the endocannabinoid 2-arachidonoylglycerol), and binding sites specific for cannabinoid (CB) receptors. The findings obtained show that nitroglycerin-induced hyperalgesia is associated with increased activity of both hydrolases and increased density of CB binding sites in the mesencephalon. In the hypothalamus we observed an increase in the activity of fatty acid amide hydrolase associated with an increase in density of CB binding sites, while in the medulla only the activity of fatty acid amide hydrolase was increased. Anandamide also proved effective in preventing nitroglycerin-induced activation (c-Fos) of neurons in the nucleus trigeminalis caudalis. These data strongly support the involvement of the endocannabinoid system in the modulation of nitroglycerin-induced hyperalgesia, and, possibly, in the pathophysiological mechanisms of migraine.