A series of triazolopurines showed structural similarity to human adenosine A(3) receptor antagonist, 9-chloro-2-(2-furanyl)-5-[(phenylacetyl)amino][1,2,4]triazolo[1,5-c]quinazoline (MRS 1220, 1). In this study, we found novel 1,2,4-triazolo[5,1-i]purine derivatives (2) showing human adenosine A(3) receptor affinities. The compounds were obtained in two steps from 5-amino-4-cyanoimidazole (33). The affinity was determined in radioligand binding assays for the cloned human adenosine A(1), A(2A), A(2B), and A(3) receptors. After the structure-activity relationship was analyzed, we determined that there was a mild parabolic relationship between the length of alkyl groups at the 5-position and the affinities at the A(3) receptor and positive correlation between the length of the substituents on phenyl groups at the 8-position and the affinities at the A(2A) receptor. These investigations led to potent and selective human adenosine A(3) receptor ligands. The most potent A(3) receptor ligand (5-n-butyl-8-(4-methoxyphenyl)-3H-[1,2,4]triazolo[5,1-i]purine (27, K(i) = 0.18 nM) and the most selective A(3) receptor ligand against A(1), A(2A), and A(2B) receptors, (5-n-butyl-8-(4-n-propoxyphenyl)-3H-[1,2,4]triazolo[5,1-i]purine (29, >19 600), were discovered.