Allosteric binding sites on the adenosine receptor family represent potential therapeutic targets for a number of conditions involving metabolic stress. This study has identified Brilliant Black BN as a novel allosteric modulator of the adenosine A1 and A3 receptors. In addition to being a food dye and pharmaceutical excipient, Brilliant Black BN is commonly used within calcium mobilization assays to quench extracellular fluorescence. Brilliant Black BN (5-500 μM) had no significant effect on the calcium mobilization stimulated by the nonselective adenosine receptor agonist 5′-(N-ethylcarboxamido)adenosine in Chinese hamster ovary cells stably transfected with the human adenosine A1 or A3 receptor. Likewise, calcium mobilization and radioligand binding assays found that Brilliant Black BN (5-500 μM) did not significantly influence the antagonism mediated by 8-cyclopentyl-1,3-dipropylxanthine (100 nM) at the A1 receptor. In contrast, the affinity of N-[9-chloro-2-(2-furanyl)[1,2,4]- triazolo[1,5-c]quinazolin-5-yl]benzene acetamide (MRS1220) at the A3 receptor and xanthine amine congener (XAC) and XAC-X-BY630 at the A1 and A3 receptors was significantly decreased in the presence of 500 μM Brilliant Black BN. A reduction in XAC potency at the A1 and A3 receptor was achieved within 1 min of Brilliant Black BN addition, despite receptors having been pre-equilibrated with antagonist. Dissociation kinetics of the fluorescent XAC derivative, XAC-XBY630, revealed that the decrease in affinity is probably due to a significant increase in dissociation rate of the antagonist in the presence of Brilliant Black BN. Taken together, these results suggest that Brilliant Black BN can act allosterically to modify ligand affinity at A1 and A3 receptors.