Extracellular adenosine mediates a multitude of biological effects, including wakefulness, antiarrythmia, bronchoconstriction and response to ischemia and oxidative stress. A family of four GPCR adenosine receptors, A1, α2A, α2B and A3, is responsible for these effects (Fredholm et al., 2001). A3, which couples to Gi/o, is expressed in mast cells along with α2B. Mice lacking A3 display reduced mast cell degranulation and bronchoconstriction in response to adenosine (Tilley et al., 2003; Zhong et al., 2003). Cloned human A3-expressing cell line is made in the Chem-3 host, which supports high levels of recombinant A3 expression on the cell surface and contains high levels of the promiscuous G protein Ga15 to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for agonists and antagonists at A3. The A3 membrane preparations are crude membrane preparations made from our proprietary stable recombinant cell lines to ensure high-level of GPCR surface expression; thus, they are ideal HTS tools for screening of antagonists of A3 interactions with its ligands. The membrane preparations exhibit a Kd of 4.7 nM for [125I]-AB-MECA. With 5mg/well A3 Membrane Prep and 5nM [125I]-AB-MECA, a greater than 3-fold signal-to-background ratio is obtained.