Adenosine A(2B) receptors, which play a role in inflammation and cancer, are of considerable interest as novel drug targets. To gain deeper insights into ligand binding and receptor activation, we exchanged amino acids predicted to be close to the binding pocket. The alanine mutants were stably expressed in CHO cells and characterized by radioligand binding and cAMP assays using three structural classes of ligands: xanthine (antagonist), adenosine, and aminopyridine derivatives (agonists). Asn282(7.45) and His280(7.43) were found to stabilize the binding site by intramolecular hydrogen bond formation as in the related A(2A) receptor subtype. Trp247(6.48), Val250(6.51), and particularly Ser279(7.42) were shown to be important for binding of nucleosidic agonists. Leu81(3.28), Asn186(5.42), and Val250(6.51) were discovered to be crucial for binding of the xanthine-derived antagonist PSB-603. Leu81(3.28), which is not conserved among adenosine receptor subtypes, may be important for the high selectivity of PSB-603. The N186(5.42)A mutant resulted in an increased potency for agonists. The interactions of the non-nucleosidic agonist BAY60-6583 were different from those of the nucleosides: while BAY60-6583 appeared not to interact with Ser279(7.42), its interactions with Trp247(6.48) and Val250(6.51) were significantly weaker compared to those of NECA. Moreover, our results discount the hypothesis of Trp247(6.48) serving as a "toogle switch" because BAY60-6583 was able to activate the corresponding mutant. This study reveals distinct interactions of structurally diverse ligands with the human A(2B) receptor and differences between closely related receptor subtypes (A(2B) and A(2A)). It will contribute to the understanding of G protein-coupled receptor function and advance A(2B) receptor ligand design.