Three structurally related non-xanthine compounds, CGS 15943, ZM 241385 and SCH 58261, are potent A2A adenosine receptor antagonists and have been used as tools in many pharmacological studies. We have now characterized their affinity and selectivity profile on human adenosine receptors stably transfected into either CHO cells (A1 and A2B receptors) or HEK-293 cells (A2A and A3 receptors). In binding studies using [3H]SCH 58261 as a radioligand, the three compounds were equally potent at A2A receptors, their K(i) value being less than 1 nM. Affinity for A1 and A3 receptors was measured using [3H]DPCPX and [125I]AB-MECA as radioligands. Given the lack of selective ligands, interaction with A2B receptors was assessed using the cAMP accumulation assay following stimulation by the adenosine receptor agonist N-ethylcarboxamidoadenosine (NECA). CGS 15943 was almost as potent at A1 receptors (K(i)3.5 nM) as at A2A receptors, showed moderate affinity for A3 receptors (K(i) 95 nM) and also interacted with A2B receptors (K(i) 44 nM; pA2 7.5). ZM 241385 showed little affinity for A1 receptors (K(i) 255 nM), and did not interact with A3 receptors (K(i)>10 microM); however, it displayed moderate affinity for A2B receptors (K(i) 50 nM; pA2 7.3). SCH 58261 had weak affinity for A1 receptors (K(i) 287 nM), no interaction with A3 receptors (K(i)>10 microM), and showed negligible interaction with A2B receptors (K(i) 5 microM; pA2 6.0). These data indicate that SCH 58261 is the most selective A2A antagonist currently available. Moreover, the different receptor selectivity of these three chemically related compounds provides useful information to progress with structure-activity relationship studies.