The 2-amino-benzoylthiophene derivative PD81,723 [(2-amino-4,5-dimethyl-trienyl)[3-(trifluoromethyl) phenyl]methanone] has been shown to allosterically enhance agonist binding and function at the adenosine A(1) receptor. The aim of the present study was to elucidate the effects of PD81,723 both as an allosteric enhancer and as an antagonist on the adenosine A(1) receptor. We investigated its effect on the human wild-type in relation to a mutant (T277A) adenosine A(1) receptor for which agonists have a greatly diminished affinity. Binding (saturation and displacement experiments) and functional adenosine 3',5'-cyclic monophosphate studies were performed, and differential effects of allosteric enhancer PD81,723 on agonists and antagonists were observed on the wild-type (wt) and mutant adenosine A(1) receptor. Our results showed opposite effects of PD81,723 on the binding of agonists and antagonists. Within the concept of a simplified two-state receptor model, it is possible that the effects of PD81,723 are mainly "allosteric", enhancing the binding of adenosine A(1) agonists and inhibiting the binding of antagonists/inverse agonists. However, the suggestion that PD81,723 acts as an allosteric inhibitor of DPCPX (1,3-dipropyl-8-cyclopentylxanthine) binding cannot be confirmed by kinetic studies, since PD81,723 does not seem to affect the dissociation kinetics of [(3)H]DPCPX. Nevertheless, our results show that the action of PD81,723 on DPCPX binding is due to more than mere competitive antagonistic activity, i.e. binding to the ligand-binding site and competing with the binding of DPCPX, as suggested previously. The effect of PD81,723 on the mutant receptor was much less pronounced. Mutation of Thr277 to Ala not only decreased agonist affinity but also inhibited the effects of PD81,723. Insensitivity of the mutT277A to PD81,723 may be linked to the fact that this mutant appears to be uncoupled from G proteins. It further supported a differential binding mode of PD81,723 compared to competitive antagonists for the adenosine A(1) receptor.