Four subtypes of adenosine receptors are currently known, that is, A(1), A(2A), A(2B), and A(3) receptors. Interestingly, quite substantial species differences exist especially between human and rat A(3) receptors. As a result, ligands such as CCPA, which are very selective for the rat A(1) receptor versus the human A(3) receptor, are substantially less selective when the human A(1) and A(3) receptors are compared. New 2-substituted and 2,N(6)-disubstituted adenosines were synthesized, and their affinities for the human adenosine A(1), A(2A), A(2B), and A(3) receptors were determined. Although large substituents on the C2-position are generally thought to yield adenosine A(2A) receptor selective ligands, the reported series of 2-triazeno-substituted adenosines had a very high affinity for the A(1) receptor. For example, 2-(3-phenylaminocarbonyltriazene-1-yl)adenosine had an affinity of 6.1 +/- 1.3 nM for the human adenosine A(1) receptor. Introduction of a diphenethyl substituent at the N(6)-position of this compound resulted in a high-affinity agonist, 3.1 +/- 0.9 nM, for the human adenosine A(1) receptor with 316- and 45-fold selectivity versus the human A(2A) and human A(3) receptors, respectively. The most selective, high-affinity human adenosine A(1) receptor agonist was the disubstituted compound N(6)-cyclopentyl-2-(3-phenylaminocarbonyltriazene-1-yl)adenosine (TCPA). TCPA had an affinity of 2.8 +/- 0.8 nM for the human adenosine A(1) receptor and was 75-fold and 214-fold selective versus the human A(2A) and human A(3) receptors, respectively. In addition, TCPA was a full agonist and inhibited the forskolin-induced cAMP production of CHO cells stably transfected with the human adenosine A(1) receptor with an IC(50) of 1.5 +/- 0.5 nM.