To provide new insights into ligand-A1 adenosine receptor (A1AR) interactions, site-directed mutagenesis was used to test the role of several residues in the first four transmembrane domains of the human A1AR. First, we replaced eight unique A1AR residues with amino acids present at corresponding transmembrane (TM) positions of A2AARs. We also tested the role of carboxamide amino acids in TMs 1-4, and the roles of Val-87, Leu-88, and Thr-91 in TM3. Following conversion of Gly-14 in TM1 to Thr-14, the affinity for adenosine agonists increased 100-fold, and after Pro-25 in TM1 was converted to Leu-25, the affinity for agonists fell. After conversion of TM3 sites Thr-91 to Ala-91, and Gln-92 to Ala-92, the affinity for N6-substituted agonists was reduced, and binding of ligands without N6 substituents was eliminated. When Leu-88 was converted to Ala-88, the binding of ligands with N6 substituents was reduced to a greater extent than ligands without N6 substituents. Following conversion of Pro-86 to Phe-86, the affinity for N6-substituted agonists was lost, and the affinity for ligands without N6 substitution was reduced. These observations strongly suggest that Thr-91 and Gln-92 in TM3 interact with the adenosine adenine moiety, and Leu-88 and Pro-86 play roles in conferring specificity for A1AR selective compounds. Using computer modeling based on the structure of rhodopsin, a revised model of adenosine-A1AR interactions is proposed with the N6-adenine position oriented toward the top of TM3 and the ribose group interacting with the bottom half of TMs 3 and 7.