A β-escin-permeabilized canine tracheal smooth muscle preparation was used to test the hypothesis that cGMP decreases Ca2+ sensitivity in airway smooth muscle primarily by inhibiting the membrane receptor-coupled mechanisms that regulate Ca2+ sensitivity and not by inhibiting Ca2+/calmodulin activation of the contractile proteins. 8-Bromo-cGMP (100 μM) had no effect on the free Ca2+ concentration-response curves generated in the absence of muscarinic receptor stimulation. In the presence of 100 μM ACh plus 10 μM GTP, 8-bromo-cGMP (100 μM) caused a rightward shift of the free Ca2+ concentration-response curve, significantly increasing the EC50 for free Ca2+ from 0.35 ± 0.03 to 0.75 ± 0.06 μM; this effect of 8- bromo-cGMP was concentration dependent from 1 to 100 μM. 8-Bromo-cGMP (100 μM) decreased the level of regulatory myosin light chain (rMLC) phosphorylation for a given cytosolic Ca2+ concentration but had no effect on the amount of isometric force produced for a given level of rMLC phosphorylation. These findings suggest that cGMP decreases Ca2+ sensitivity in canine tracheal smooth muscle primarily by inhibiting the membrane receptor-coupled mechanisms that modulate the relationship between cytosolic Ca2+ concentration and rMLC phosphorylation.