A₁adenosine receptor antagonists have been proposed to possess an interesting range of potential therapeutic applications. We have already reported the synthesis and the biological characterization of a family of pyrazolo[3,4-b]pyridine derivatives as A₁adenosine ligands endowed with an antagonistic profile. In the present work, we report the LC separation of enantiomers of our most active A₁antagonists together with the determination of their absolute configuration by means of X-ray crystal structure analysis. Biological assays confirmed a different activity for the two enantiomers, with the R one showing the higher human A₁AR affinity. We also developed a homology model of this receptor subtype in order to suggest a binding disposition of the ligands into the hA₁AR. All of the obtained data suggest that the compound's chirality plays a key role in A₁ affinity.