Studies of endogenous cannabinoid agonists, such as 2-arachidonylglycerol (2-AG), have revealed their potential to exert modulatory actions on other receptor systems in addition to their ability to activate cannabinoid receptors. This study investigated the effect of cannabinoid ligands on the human adenosine A 3 (hA 3R) receptor. The endocannabinoid 2-AG was able to inhibit agonist ([^1^2^5I]N^6-(4-amino-3-iodobenzyl) adenosine-5*-(N-methyluronamide) - [^1^2^5I] AB MECA) binding at the hA 3R. This inhibition occurred over a narrow range of ligand concentration and was characterized by high Hill coefficients suggesting a non-competitive interaction. Furthermore, in the presence of 2-AG, the rate of [^1^2^5I] AB MECA dissociation was increased, consistent with an action as a negative allosteric modulator of the hA 3R. Moreover, by measuring intracellular cAMP levels, we demonstrate that 2-AG decreases both the potency of an agonist at the hA 3R and the basal signalling of this receptor. Since the hA 3R has been shown to be expressed in astrocytes and microglia, these findings may be particularly relevant in certain pathological states such as cerebral ischemia where levels of 2-AG and anandamide are raised.