A wide variety of plants produce compounds that contain the 4-hydroxy-3-methoxybenzyl or "vanillyl" group. These vanilloid compounds include such well-known food molecules as vanillin, capsaicin, gingerol, and curcumin. Vanilloid compounds have been widely studied for their medicinal properties, and one property that most of them have in common is the ability to induce analgesia. Studies have indicated multiple potential cellular mechanisms for this analgesic activity, including TRPV1 receptors, opioid receptors, and adenosine receptors (ARs). There are only a few studies on the interaction of vanilloid compounds with ARs, and to our knowledge no previous study has measured the binding affinity of any vanilloid compound with either of the two AR subtypes that are linked to the inhibitory G subunit (subtypes A and A ). Our study objective was to assess the binding affinity and receptor activation of a novel curcumin isomer, cis-transcurcumin (CTCUR), at AR subtypes A and A . Since agonism of these subtypes is associated with analgesia, we hypothesized that CTCUR would be an agonist at AR subtypes A and A . Two lines of Chinese hamster ovary cells were used, each transfected to overexpress one of the AR subtypes. Cell survival assays were performed to assess toxicity. Competitive binding assays and confocal microscopy were performed to measure binding affinity. Computer docking was also performed, to assess location of binding. Assays of intracellular cAMP levels were performed to measure receptor activation. Survival results indicate that CTCUR is not toxic to CHO cells at any of the concentrations tested (the highest being 100 μM). Competition assay results indicate that CTCUR binds to subtype A with a K of 306 nM and to subtype A with a K of 400 nM. Microscopy results confirm sub-micromolar affinity for A , but not for A . Docking results confirm sub-micromolar affinity for both subtypes, and furthermore indicate that CTCUR binds to the "toggle switch" domain of α helix 6 that is important for receptor activation. Intracellular cAMP results indicate that CTCUR acts as an agonist of ARs. Thus, we fail to reject our hypothesis. These results are of interest because they provide the first in vitro confirmation of the hypothesis that vanilloid compounds can activate ARs, in accordance with the results of previous studies in vivo. Vanilloid compounds that activate ARs are therefore worthy of further study, as a potential class of analgesic therapeutics.