Until recently, the characterization of adenosine A(2B) receptors has been hampered by the lack of high affinity radioligands. This study describes the synthesis and in vitro characterization of the radiolabeled derivative of OSIP339391, a novel, potent, and selective pyrrolopyrimidine A(2B) antagonist. OSIP339391 had a selectivity of greater than 70-fold for A(2B) receptors over other human adenosine receptor subtypes. The radiolabel was introduced by hydrogenation of the acetylenic precursor with tritium gas resulting in the incorporation (on average) of three tritium atoms in the molecule, yielding a ligand with specific activity of 87Ci/mmol (3.2TBq/mmol). Using membranes from HEK-293 cells expressing the human recombinant A(2B) receptor, [3H]OSIP339391 was characterized in kinetic, saturation, and competition binding experiments. From the association and dissociation rate studies, the affinity was 0.41nM and in close agreement with that found in saturation binding experiments (0.17nM). In competition, binding studies using 0.5nM [3H]OSIP339391, the affinity of a range of agonists and antagonists was consistent with previously reported data. Thus, [3H]OSIP339391 is a novel, selective, and high affinity radioligand that can be a useful tool in the further exploration and characterization of recombinant and endogenous adenosine A(2B) receptors.