Stimulation of calcium-activated potassium (KCa) channels in airway smooth muscle cells by phosphorylation-dependent and membrane-delimited, G protein actions has been reported (Kume, H. A. Takai, H. Tokuno, and T. Tomita. 1989. Nature [Lond.]. 341:152-154; Kume, H., M. P. Graziano, and M. I. Kotlikoff. 1992. Proc. Natl. Acad. Sci. USA. 89:11051-11055). We show that beta-adrenergic receptor/channel coupling is not affected by inhibition of endogenous ATP, and that activation of KCa channels is stimulated by both alpha S and cAMP-dependent protein kinase (PKA). PKA stimulated channel activity in a dose-dependent fashion with an EC50 of 0.12 U/ml and maximum stimulation of 7.38 +/- 2.04-fold. Application of alpha S to patches near maximally stimulated by PKA significantly increased channel activity to 15.1 +/- 3.65-fold above baseline, providing further evidence for dual regulatory mechanisms and suggesting that the stimulatory actions are independent. Analysis of channel open-time kinetics indicated that isoproterenol and alpha S stimulation of channel activity primarily increased the proportion of longer duration events, whereas PKA stimulation had little effect on the proportion of short and long duration events, but resulted in a significant increase in the duration of the long open-state. cAMP formation during equivalent relaxation of precontracted muscle strips by isoproterenol and forskolin resulted in significantly less cAMP formation by isoproterenol than by forskolin, suggesting that the degree of activation of PKA is not the only determinant of tissue relaxation. We conclude that beta-adrenergic stimulation of KCa channel activity and relaxation of tone in airway smooth muscle occurs, in part, by means independent of cyclic AMP formation.