KCNQ (Kv7 family) potassium (K⁺) channels were recently found in airway smooth muscle cells (ASMCs) from rodent and human bronchioles. In the present study we evaluated KCNQ channel expression and their role in constriction/relaxation of rat airways. Real time RT-PCR analysis revealed expression of KCNQ4 > KCNQ5 > KCNQ1 > KCNQ2 > KCNQ3 and patch clamp electrophysiology detected KCNQ currents in rat ASMCs. In precision-cut lung slices (PCLS), the KCNQ channel activator retigabine induced a concentration-dependent relaxation of small bronchioles pre-constricted with methacholine (EC₅₀ = 3.6 ± 0.3 µM). Bronchoconstriction was also attenuated in the presence of two other structurally unrelated KCNQ channel activators: zinc pyrithione (1 μM; 22 ± 7%), and 2,5-dimethylcelecoxib (10 μM; 24 ± 8%). The same three KCNQ channel activators increased KCNQ currents in ASMCs by 2- to 3-fold. The bronchorelaxant effects of retigabine and zinc pyrithione were prevented by inclusion of the KCNQ channel blocker XE991. A long-acting β₂-adrenergic receptor agonist, formoterol (10 nM), did not increase KCNQ current amplitude in ASMCs, but formoterol (1-1000 nM) did induce a time- and concentration-dependent relaxation of rat airways, with a notable desensitization during a 30 min treatment or with repetitive treatments. Co-administration of retigabine (10 μM) with formoterol produced a greater peak and sustained reduction of methacholine-induced bronchoconstriction, and reduced the apparent desensitization observed with formoterol alone. Our findings support a role for KCNQ K⁺ channels in the regulation of airway diameter. Combination of a β₂-adrenergic receptor agonist with a KCNQ channel activator may improve bronchodilator therapy.