Suramin analogues act as direct antagonists of heterotrimeric G proteins because they block the rate-limiting step of G protein activation (i.e., the dissociation of GDP prebound to the G protein alpha subunit). We have used the human brain A1 adenosine receptor and the rat striatal D2 dopamine receptor, two prototypical Gi/G(o)-coupled receptors, as a model system to test whether the following analogues suppress the receptor-dependent activation of G proteins: 8-(3-nitrobenzamido)-1,3,5-naphthalenetrisulfonic acid (NF007), 8-(3-(3-nitrobenzamido)-benzamido)-1,3,5-naphthalenetrisulfonic acid (NF018); 8,8'-(carbonylbis(imino-3,1-phenylene))bis-(1,3,5-naphthalenetr isulfonic acid) (NF023); 8,8'-(carbonylbis(imino-3,1-phenylene)carbonylimino-(3,1- phenylene)) bis(1,3,5-naphthalenetrisulfonic acid) (NF037); and suramin. Suramin and its analogues inhibit the formation of the agonist-specific ternary complex (agonist/receptor/G protein). This inhibition is (i) quasicompetitive with respect to agonist binding in that it can be overcome by increasing receptor occupancy but (ii) does not result from an interaction of the analogues with the ligand binding pocket of the receptors because the binding of antagonists or of agonists in the absence of functional receptor/G protein interaction is not affected. In addition to suppressing the spontaneous release of GDP from defined G protein alpha subunits, suramin and its analogues reduce receptor-catalyzed guanine nucleotide exchange. The site, to which suramin analogues bind, overlaps with the docking site for the receptor on the G protein alpha subunit. The structure-activity relationships for inhibition of agonist binding to the A1 adenosine receptor (suramin > NF037 > NF023) and of agonist binding to the inhibition D2 dopamine receptor (suramin = NF037 > NF023 > NF018) differ. Thus, NF037 discriminates between the ternary complexes formed by the agonist-liganded D2 dopamine receptors and those formed by the A1 adenosine receptor with > 10-fold selectivity. Therefore, our results also show that inhibitors can be identified that selectively uncouple specific receptor/G protein tandems.