AbstractType 1 cannabinoid receptors (CB1R) are G‐protein‐coupled receptors that mediate several actions of the endocannabinoid anandamide (N‐arachidonoylethanolamine; AEA) in the central nervous system. Here we show that cholesterol enrichment of rat C6 glioma cell membranes reduces by approximately twofold the binding efficiency (i.e., the ratio between maximum binding and dissociation constant) of CB1R and that activation of CB1R by AEA leads to approximately twofold lower [35S]GTPγS binding in cholesterol‐treated cells than in controls. In addition, we show that CB1R‐dependent signaling via adenylate cyclase and p42/p44 mitogen‐activated protein kinase is almost halved by cholesterol enrichment. Unlike CB1R, the other AEA‐binding receptor TRPV1, the AEA synthetase NAPE‐PLD, and the AEA hydrolase FAAH are not modulated by cholesterol, whereas the catalytic efficiency (i.e., the ratio between maximal velocity and Michaelis‐Menten constant) of the AEA membrane transporter AMT is almost doubled compared with control cells. These data demonstrate that, among the proteins of the “endocannabinoid system,” only CB1R and AMT critically depend on membrane cholesterol content. This observation may have important implications for the role of CB1R in protecting nerve cells against (endo)cannabinoid‐induced apoptosis. © 2005 Wiley‐Liss, Inc.