We have examined the effect of increased Gs protein levels on the abilities of three different beta2-agonists to induce GTP shifts and stimulate adenylyl cyclase response in an effort to investigate the kinetic association between the beta2-adrenergic receptor Gs and adenylyl cyclase. Agonist competition binding analysis and adenylyl cyclase concentration-response assays revealed that increases in Gs protein resulted in proportional increases in the areas of the GTP shift and adenylyl cyclase activity. Changes in the magnitude of the GTP shift were evaluated with a novel and straightforward approach for analyzing the GTP shift data that allowed us to determine the proportion of high agonist affinity binding receptor population and the apparent dissociation constant between the agonist bound receptor and Gs, regardless of the Gs protein level or the type of beta2-agonist. Using this method, we concluded that increased Gs results in the accumulation of the receptor population displaying high affinity towards agonist (HRGs) by increasing the number of receptor-Gs complexes (to a receptor:Gs protein ratio of about 0.7 at maximal Gs expression) without affecting the affinity between hormone bound receptor and Gs. Using the Gs protein levels determined with our novel analysis, we ran simulations using the theoretical shuttle model equation that relates the EC50 to available Gs. Fitting the simulations to experimental data required a receptor to catalytic unit ratio of 0.45 and revealed at least two distinct stages for beta2-agonist-stimulated adenylyl cyclase activity, namely, the activation of Gs by the beta2-adrenergic receptor (a step whose rate is dependent on the type of agonist used to stimulate activity), and the activation of adenylyl cyclase by active Gs (a step whose rate is independent of the type of agonist).