A new series of pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines bearing various substituents at both the N5-pyrimidinyl and N8-pyrazolyl positions have been synthesized, and their binding affinities at the four human adenosine receptor subtypes (hA(1), hA(2A), hA(2B), and hA(3)) have been evaluated. All the described compounds contain arylacetyl moieties at the N5 position and arylalkyl substituents at the N8 position. Surprisingly, all the compounds present their most potent affinities at the hA(2B) adenosine receptor with a range of selectivities against the other subtypes. When bulky groups are present simultaneously at the N5 and N8 positions (e.g., compound 9), the best selectivity for the hA(2B) receptor was observed (K(i)(hA(1)) = 1100 nM; K(i)(hA(2A)) = 800 nM; K(i)(hA(2B)) = 20 nM; K(i)(hA(3)) = 300 nM, K(i)(hA(1)/A(2B)) = 55, K(i)(hA(2A)/A(2B)) = 40, K(i)(hA(3)/hA(2B)) = 15). To understand the molecular significance of these results, we compared the putative TM (transmembrane) binding motif of compound 9 on both hA(2B) and hA(3) receptors. From our docking studies, compound 9 fits neatly inside the TM region of the hA(2B) receptor but not in the corresponding hA(3) region, illustrating significant differences between the two subtypes. The study herein presented permits an understanding of why the bioisosteric replacement of an -NH, present in previously reported hA(3) receptor antagonists, with a -CH(2) group at the N5 position induces such large differences in hA(2B)/hA(3) affinity. In the molecular structure of the hA(3) receptor, two residues, Ser243 (TM6) and Ser271 (TM7), create a hydrophilic region, which seems to permit a better accommodation of the phenylurea series into this putative hA(3) binding site than the phenylacetyl series.