Receptors and heterotrimeric G-proteins interact with a high degree of specificity, the molecular basis of which is only partially understood. In the present study, we analyzed the influence of different G-protein betagamma-subunits on the coupling of the beta2-adrenergic receptor to G(s). Sf9-cells were infected with baculoviruses coding for the beta2-adrenergic receptor, alpha(s,Short) or alpha(s,Long), and various beta- and gamma-subunits. The ability of different beta- and gamma-subunits to correctly dimerize was assessed by limited proteolysis of proteins expressed in Sf9-cells and additionally by analysis of beta/gamma-interaction in the yeast two-hybrid system. Agonist-induced GTPgammaS-binding to alpha(s,Short)beta(1)gamma-trimers was significantly higher than to alpha(s,Short)beta2gamma-combinations, when gamma4, gamma5, or gamma7 were co-expressed. Because beta(5) did not support coupling of the beta(2)-adrenergic receptor to G(s), the 87 C-terminal amino acids of Gbeta(5) assumed to encompass the beta-subunit interface with the receptor were substituted by the corresponding sequence of beta(1). Whereas this beta(5)/beta(1)-chimera did not promote GTPgammaS-binding to alpha(s), histamine H(1)-receptor-dependent GTPgammaS-binding to alpha(q) was supported by this chimeric beta-subunit and by wild-type beta(5). Our findings argue that the betagamma-subunit composition contributes directly to the specificity of beta(2)-adrenergic receptor-mediated G(s)-activation.