To provide new insights into the regions of the human A1 adenosine receptor (A1AR) involved in ligand binding, a series of chimeric human A1 and rat A2a adenosine receptors (A1/A2a) were studied. Binding studies were initially performed on acutely transfected COS cells using fixed doses of the A2aAR agonist [3H]CGS-21680, the A1AR agonist [3H]2-chloro-N6-cyclopentyladenosine (CCPA), and the A1AR antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine (DPCPX). When the region of the A2aAR from the amino terminus to the end of transmembrane (TM) 1 was replaced by the corresponding region of the A1AR (A1TM1/A2a), [3H]CGS-21680 and [3H]CCPA binding was detectable. When an A1TM1-2/A2a construct was studied, [3H]CGS-21680 binding was lost and [3H] DPCPX binding appeared. Saturation studies using [3H]CCPA revealed that the A1TM1/A2a construct had low affinity. However, with the subsequent addition of A1AR TMs 2-4 receptor affinity improved markedly. Saturation studies using [3H]DPCPX also revealed that the TMs 1-4 of the A1AR conferred wild-type receptor affinity. When the ligand binding properties of A1TM1-4/A2a, A1TM1-6/A2a, and wild type A1AR constructs were directly compared, no differences were found using 10 different compounds. When truncated A1ARs that extended from the amino terminus to shortly after TM4 were examined, no binding was detectable suggesting that the amino half of the receptor alone is not sufficient for ligand binding. Collectively, these data suggest that the important determinants for A1AR agonist and antagonist binding and ligand specificity are present in TMs 1-4.