2-Amino-3-benzoylthiophenes such as PD 81,273 (PD) have been shown to increase agonist, but not antagonist, radioligand binding and to enhance functional effects of A1 adenosine receptor (A1AR) activation in tissues derived from rats and guinea-pigs. The mechanism by which PD produces this allosteric enhancement, and its effect on human A1ARs was not known. In this study, we demonstrate that PD modifies recombinant human A1AR binding and function in stably transfected CHO cells. In membranes, PD (20 microM) causes: (i) a 3-fold increase in the fraction of receptors found in a high affinity G-protein coupled conformation as assessed by the binding of [125I]N6-(3-iodo-4-aminobenzyladenosine) (125I-ABA), an A1AR agonist; (ii) a 2.44-fold increase in the potency of the agonist R-N6-phenylisopropyladenosine (R-PIA) to compete for binding with the antagonist radioligand, [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]CPX); (iii) a 1.5-fold increase in the t1/2 of 125I-ABA to dissociate from A1AR; and (i.v.) a 2.2-fold increase in the concentration of guanosine-5'-3-O-(thio)triphosphate (GTP gamma S) required to half-maximally uncouple receptor-G-protein complexes. In intact CHO cells expressing A1AR, PD increases the potency of R-PIA to decrease forskolin-stimulated cAMP accumulation by 3.3-fold. We speculate that PD binds to A1AR at a site distinct from the agonist binding site and stabilizes agonist-R-G complexes.