Contradictory results exist concerning the effects of systemic injections of CB(1) cannabinoid receptor agonists on anxiety-related behaviors. Direct drug administration into brain structures related to aversive responses can potentially help to clarify the role of cannabinoids on anxiety. One such structure is the midbrain dorsolateral periaqueductal gray (dlPAG). Therefore, the aim of this study was to test the hypothesis that the activation of the CB(1) receptor in the dlPAG would attenuate anxiety-related behaviors. Male Wistar rats with cannula aimed at the dlPAG received injections of the endogenous cannabinoid anandamide, the anandamide transport inhibitor AM404, the anandamide analogue ACEA or the CB(1) receptor antagonist AM251, and were submitted to the elevated plus maze (EPM), an animal model of anxiety. Anandamide (0.5-50pmol) and ACEA (0.05-5pmol) induced anxiolytic-like effects with bell-shaped dose-response curves, the higher doses being ineffective. The anandamide anxiolytic effect was potentiated by AM404 (50pmol) and prevented by AM251 (100pmol). Neither AM404 (0.5-50pmol) nor AM251 (1-100pmol) alone modified the animal behavior in the EPM. The present study suggests that the dlPAG is a possible neuroanatomical site for anxiolytic-like effects mediated by CB(1) agonists. Furthermore, this work supports the importance of neuronal uptake as a mechanism that limits the in vivo actions of anandamide.