Inhaled beta-agonists are effective at reversing bronchoconstriction in asthma but exhibit significant side effects including tachyphylaxis and possibly increased asthma-related mortality. The mechanisms by which beta-agonists exert both their beneficial and detrimental effects are controversial and poorly understood. The cAMP-dependent protein kinase (PKA) is the historically accepted effector of beta-agonist-mediated bronchorelaxation, though this assumption is based on associative and not direct evidence. Recent studies have asserted that exchange protein activated by cAMP (Epac)--and not PKA--mediates the relaxation of ASM observed with beta-agonist treatment. This study aims to clarify the role of PKA in the pro-relaxant effects of beta-agonists on airway smooth muscle as well as the ability of the kinase to exert negative feedback on this signaling. Inhibition of PKA activity via expression of the PKI and RevAB peptides results in increased beta-agonist-mediated cAMP release, abolishes the inhibitory effect of isoproterenol on intracellular calcium flux, and significantly attenuates histamine-stimulated MLC-20 phosphorylation. Analyses of ASM cell and tissue contraction demonstrate that PKA inhibition eliminates most, if not all, beta-agonist-mediated relaxation of contracted smooth muscle. These findings demonstrate that PKA is the predominant and physiologically relevant effector through which beta-agonists exert their bronchorelaxant effects. To investigate compartmentalized cAMP and PKA signaling and regulation related to beta-agonists, analyses of mRNA and protein expression identified 11 A-kinase anchoring proteins (AKAPs) in human ASM. Disruption of AKAP-PKA interactions via peptides AKAP-IS or Ht31 has minimal effects on whole cell cAMP signaling stimulated by beta-agonist, but significantly increases the duration of plasma membrane-delineated cAMP. Thus PKA, in addition to its effector function, significantly contributes to homologous desensitization of the beta-2-AR in ASM through mechanisms dependent on its localization near the plasma membrane. While the role of specific AKAPs in ASM remains undefined, these findings clearly demonstrate the importance of targeted PKA activity in beta-agonist-mediated signaling. Specific manipulation of PKA activity or AKAP complexes may provide a means to improve upon current bronchodilator therapies for the treatment of asthma.