Endocannabinoids (ECs) are important neuromodulators involved in a plethora of physiological processes such as modulation of synaptic transmission, neuroprotection, immune function, and neurodevelopment, among others. However, still lacking is a detailed study on the presence of this system in the circumventricular areas, brain structures controlling the interaction between cerebrospinal fluid and brain parenchyma. The aim of this work was to provide the anatomical basis supporting a functional role of ECs in the activity of circumventricular areas. To this end, an immunohistochemical study of the EC system in rat brain was performed. Receptors and synthesizing and degrading enzymes for ECs were widely distributed in rat ependyma and subependyma, marginal glia, and circumventricular organs (CVOs) such as the choroid plexus, subfornical organ, subcommissural organ, median eminence, and area postrema. These zones constitute barrier systems between the brain parenchyma and the ventricular or subarachnoid cerebrospinal fluid (CSF) and between the extracellular hemal milieu of CVOs and the brain parenchyma or the CSF. By immunohistochemistry and real-time polymerase chain reaction we found DAGLα, DAGLβ, NAPE-PLD, MAGL, and FAAH in the ependyma. These finding suggest that the ependyma can release and clear ECs from the ventricular CSF. Subependymal astrocytes and tanycytes displayed DAGLα immunoreactivity but parenchymal astrocytes did not express EC-synthesizing enzymes, thus establishing a sharp distinction between these two astrocyte populations. CB1 was located in fibers innervating discrete subventricular zones such as the neurogenic striatal subventricular zone and the fourth ventricle. CB1 fibers also innervated some CVOs. J. Comp. Neurol. 518:3065–3085, 2010. © 2010 Wiley-Liss, Inc.