The human beta1-adrenoceptor (beta1AR) and beta2-adrenoceptor (beta2AR) couple to Gs-proteins to activate adenylyl cyclase (AC). There are differences in desensitization between the beta2AR and the originally cloned Gly389-beta1AR, but with respect to ternary complex formation, constitutive activity, and AC activation the picture is unclear. To learn more about the similarities and differences between the beta1AR and beta2AR, we analyzed coupling of the Gly389-beta1AR to the G(s(alpha)) splice variants Gs(alpha)L and Gs(alpha)S using beta1AR-Gs(alpha) fusion proteins expressed in Sf9 cells and compared the data with previously published data on beta2AR-Gs(alpha) fusion proteins (Seifert et al., J Biol Chem 1998;273:5109-16). Fusion ensures defined receptor/G-protein stoichiometry and efficient coupling. The agonist (-)-isoproterenol stabilized the ternary complex at beta1AR-Gs(alpha)S, beta1AR-Gs(alpha)L, beta2AR-Gs(alpha)S, and beta2AR-Gs(alpha)L with similar efficiency. beta1AR-Gs(alpha)L but not beta1AR-Gs(alpha)S showed the hallmarks of constitutive activity as assessed by increased potencies and efficacies of partial agonists and AC activation by the agonist-free receptor. Similar differences were observed previously for beta2AR-Gs(alpha)S and beta2AR-Gs(alpha)L. beta1AR-Gs(alpha)S and beta2AR-Gs(alpha)S were similarly efficient at activating AC, but beta1AR-Gs(alpha)L was approximately 4-fold more efficient at activating AC than beta2AR-Gs(alpha)L. Our data show that (i) the beta1AR and beta2AR are similarly efficient at stabilizing the ternary complex with Gs(alpha) splice variants, (ii) Gs(alpha)L confers constitutive activity to the beta1AR and beta2AR, and (iii) the beta1AR coupled to Gs(alpha)L is more efficient at activating AC than the beta2AR coupled to Gs(alpha)L. These data help us understand some of the discrepancies regarding similarities and differences between the beta1AR and beta2AR.