Previous studies in our laboratory identified N6-endonorbornyl-9-methyladenine (N-0861) as a highly selective (100-fold) A1-adenosine receptor antagonist (KB = 500 nM). However, its moderate potency limits the degree of A1-receptor blockade that can be achieved by systemically administered N-0861. Structure activity studies were undertaken to invent a compound that had greater affinity for the A1-adenosine receptors than N-0861. C8-N-methylisopropylamino-N6-5'-endohydroxy-N-0861 (WRC-0571) inhibited [3H]-N6-cyclohexyladenosine (CHA) binding to guinea pig A1-receptors with a Ki value of 1.1 nM. WRC-0571 was 200-fold less potent at inhibiting [3H]-5'-N-ethylcarboxamidoadenosine binding to bovine A2a receptors (Ki = 234 nM). WRC-0571 also inhibited the binding of radioligands to cloned human A1, A2a and A3 adenosine receptors with affinities of 1.7, 105 and 7940 nM, respectively. Thus in human adenosine receptors, WRC-0571 is 62-fold selective for the A1 vs. A2a and 4670-fold selective for the A1 vs. A3 receptors; WRC-0571 is therefore the most A1 vs. A3 selective compound yet described. In guinea pig isolated atria, WRC-0571 antagonized the A1-mediated negative inotropic responses to 5'-N-ethylcarboxamidoadenosine (NECA) with a KB of 3.4 nM. WRC-0571 was more than 2500-fold less potent at antagonizing NECA-induced A2b-mediated relaxation in guinea pig aorta. In anesthetized rats WRC-0571 antagonized adenosine-induced bradycardia at concentrations as low as 1 nmol/kg but failed to antagonize A2-mediated hindquarter vasodilation at concentrations up to 10,000 nmol/kg. WRC-0571 is orally active at concentrations as low as 0.3 mumol/kg. WRC-0571 is therefore a highly potent, highly selective antagonist of A1-adenosine receptors both in vitro and in vivo.