Myosin light chain kinase from smooth muscle has been shown to be phosphorylated by cyclic AMP-dependent protein kinase, which leads to a decrease in the affinity of the kinase for Ca2+ . calmodulin and, hence, a decrease in enzymatic activity. This event has been proposed as a mechanism for the relaxation of smooth muscle in response to increased intracellular concentrations of cyclic AMP. The ratio of myosin light chain kinase activities measured in the presence of 4 microM or 100 microM Ca2+, at 1 microM calmodulin, permits evaluation of such a change in the calmodulin activation properties of myosin light chain kinase. This activity ratio was decreased by phosphorylation of either purified bovine tracheal smooth muscle myosin light chain kinase, or the endogenous myosin light chain kinase in a homogenate of tracheal smooth muscle, with the addition of the catalytic subunit of cyclic AMP-dependent protein kinase. The ratio was unchanged, however, by activation of the endogenous cyclic AMP-dependent protein kinase in homogenates of tracheal smooth muscle by the addition of cyclic AMP. Incubation of tracheal smooth muscle with isoproterenol, at a concentration sufficient to relax the muscle and to increase phosphorylase a formation, had no effect upon the activity ratio. Incubation of tracheal smooth muscle for 2 hr in the presence of carbachol resulted in a transient increase and then a decrease in myosin light chain phosphate content to control values with no decrease in isometric force. The addition of isoproterenol at 2 hr still resulted in relaxation. These findings are inconsistent with a role of myosin light chain kinase phosphorylation in mediating relaxation of tracheal smooth muscle by beta-adrenergic agonists. Cyclic AMP-dependent effects on cytoplasmic calcium concentrations may be more important in mediating relaxation.