Abstract It has been suggested that endocannabinoids, anandamide (AEA) and 2-arachidonoyl glycerol (2-AG), might be the promising anti-cancer agents in clinical fields of cancer treatment. In this study, we tried to check the anti-cancer effects of AEA and 2-AG in head and neck squamous cell carcinoma (HNSCC) cell lines. AEA inhibited effectively cell proliferation of three HNSCC cells (SNU-1041, 1066 and 1076) but 2-AG did not. Cannabinoid receptor-1 (CB1) was expressed only in SNU-1066 and expression of VR1 was observed in all tested cells (no CB2 was detected). Anti-cancer effect of AEA seemed to be mediated by their receptors-independent action since the antagonist of CB1 and VR1 (AM251, cay10448 and capsazepine) did not reverse AEA-inhibited cell proliferation. From references on cancer-cell killing effect of COX-2 metabolites of AEA in some types of cancer such as colon cancer, we checked the possibility of COX-2-mediated anti-cancer effect of AEA in HNSCC cells but COX-2 inhibition (by its inhibitors and siRNA) had no effect on it. Instead, we observed the increase of reactive oxygen species (ROS) and 8-isoprostane production by AEA and antioxidants (NAC and ebselen) reversed AEA-inhibited cell proliferation partially. In addition, AM404 and cay10412 (inhibitors of AEA transporter) reversed perfectly AEA-inhibited cell proliferation. These findings suggest that AEA might have anti-cancer effect by their receptors-independent action in intracellular localization of HNSCC cells (such as increase of ROS). The investigation on detailed mechanism of anti-cancer action of AEA in HNSCC cells is ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3811. doi:1538-7445.AM2012-3811