Adenosine A3 receptors are promising drug targets for a number of conditions like inflammatory diseases including asthma, ischemic injury or certain types of cancer. Consequently, intense efforts are dedicated to the development of selective A3 agonists and antagonists. The only tritiated agonist that is available for radioligand binding is the nonselective [3H]5′-N-ethylcarboxamidoadenosine ([3H]NECA). Based on a recently characterized series of 2-substituted adenosine receptor agonists we developed a tritiated selective A3 radioligand with high affinity. From this series 2-hexyn-1-yl-N6-methyladenosine (HEMADO) with a Ki-value of 1.1 nM at the human A3 subtype was chosen. HEMADO is 300-fold selective versus the A1 subtype, and 1100-fold and more than 25,000-fold selective compared to the adenosine A2A and A2B receptors, respectively. The tritiated derivative [3H]HEMADO exhibited the same affinity as the unlabeled precursor. In concentrations up to 10 nM no specific binding to adenosine A1, A2A or A2B receptors was observed confirming the high selectivity for adenosine A3 receptors. Characterization of [3H]HEMADO in radioligand binding studies revealed reversible binding to the human adenosine A3 subtype. In saturation binding studies for the A3 subtype a KD-value of 1.1 nM was determined. Nonspecific binding at a radioligand concentration of 1 nM amounted to 1–2% of total binding. Competition binding with a panel of adenosine receptor ligands clearly confirmed the correct A3 pharmacology of the binding site labeled by [3H]HEMADO. With [3H]HEMADO we present a tritiated agonist with high affinity and A3-selectivity and very low nonspecific binding. [3H]HEMADO is a useful tool for specific screening for A3 receptor agonists and antagonists in improved radioligand binding assays with the human subtype.