Cell membranes expressing the human A 2A receptor were incubated with [ 3 H]-ZM24385 in assay buffer (50 mM HEPES, pH 7.4) in a total assay volume of 200 µl with a final DMSO concentration of 1%. After 120 minutes incubation at room temperature the reaction was terminated by rapid filtration through GF/C 96-well glass fibre plates with 5 x 0.25 ml washes with ddH 2 O using a Tomtec cell harvester. Bound radioactivity was determined through liquid scintillation using Lablogic SafeScint and detected on a MicroBeta liquid scintillation counter. Non-specific binding was determined as that remaining in the presence of a 1 μM saturating concentration of the antagonist CGS15943. Competition binding was performed as above by incubating membranes with 0.5 nM concentration of [ 3 H]-ZM24385 and a range of concentrations of the test compound (10 point concentration curves at half log intervals). IC 50 values were derived from fitting to a four parameter logistic equation in PRISM (GraphPad Software, San Diego, CA, USA). Apparent Ki values were derived using the equation of Cheng and Prusoff .