We studied fusion proteins between the human adenosine A1 receptor and different 351Cys-mutated Gi1 α-subunits (A1-Giα) with respect to two important concepts in receptor pharmacology, i.e. allosteric modulation and constitutive activity/inverse agonism. The aim of our study was twofold. We first analysed whether such fusion products are still subject to allosteric modulation, and, secondly, we investigated the potential utility of the fusion proteins to study constitutive receptor activity. We determined the pharmacological profile of nine different A1-Giα fusion proteins in radioligand binding studies. In addition, we performed [35S]GTPγS binding experiments to study receptor and G protein activation of selected A 1-Giα fusion proteins. Compared to unfused adenosine A1 receptors, the affinity of N6-cyclopentyladenosine (CPA) at wild-type A1-Giα fusion proteins ( 351Cys) increased more than eightfold, while the affinity of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) did not change significantly. Furthermore, we showed that the allosteric enhancer of agonist binding, PD81,723 (2-amino-4,5-dimethyl-3-thienyl-[3-(trifluoromethyl)-phenyl]methanone), elicited similar effects on ligand binding; i.e. CPA binding to the A 1-Giα fusion proteins was enhanced, whereas the affinity of DPCPX was hardly affected. Moreover, sodium ions were unable to decrease agonist binding to the majority of the A1-G iα fusion proteins, presumably because they exhibit their effect through uncoupling of the R-G complex. From [35S]GTPγS binding experiments, we learned that all the A1-Giα fusion proteins tested had a higher basal receptor activity than the unfused adenosine A1 receptor, thereby providing improved conditions to observe inverse agonism. Moreover, the maximal CPA-induced stimulation of basal [35S]GTPγS binding was increased for the five A 1-Giα fusion proteins tested, whereas the inhibition induced by 8-cyclopentyltheophylline (CPT) was more pronounced at 351Cys, 351Ile, and 351Val A1-G iα fusion proteins. Thus, the maximal receptor (de)activation depended on the amino acid at position 351 of the Gi α-subunit. In conclusion, A1-Giα fusion proteins, especially with 351Cys and 351Ile, can be used as research tools to investigate inverse agonism, due to their increased readout window in [ 35S]GTPγS binding experiments. © 2004 Elsevier B.V. All rights reserved.