Heterotrimeric G proteins are essential mediators of intracellular signaling of G protein-coupled receptors. The Gq/11subfamily consists of Gq, G11, G14 and G16 proteins of which all but G16 are inhibited by the structurally related natural products YM-254890 and FR900359. These inhibitors act by preventing the GDP/GTP exchange, which is necessary for activation of all G proteins. A homologous putative binding site for YM-254890/FR900359 can also be found in members of the other three G protein families; Gs, Gi/o and G12/13, but none of the published analogs of YM-254890/FR900359 have shown any inhibitory activity for any of these. To explain why the YM-254890/FR900359 scaffold only inhibits Gq/11/14, the present study delineated the molecular selectivity determinants by exchanging amino acid residues in the YM-254890/FR900359 binding site in Gq and Gs We found that the activity of a Gs mutant with a Gq-like binding site for YM-254890/FR900359 can be inhibited by FR900359 and a minimum of three mutations are necessary to introduce inhibition in Gs In all, this suggest that although the YM-254890/FR900359 scaffold has proven unsuccessful to derive Gs, Gi/o and G12/13 inhibitors, the mechanism of inhibition between families of G proteins is conserved opening up for targeting by other, novel inhibitor scaffolds. In lack of a selective Gαs inhibitor, FR900359 sensitive Gas mutants may prove useful in studies where delicate control over Gαs signaling would be of the essence.