The unique large-conductance voltage- and calcium-activated potassium (BK) channel is highly expressed in respiratory tract smooth muscle cells but its physiological role for airway function is ill-defined. Therefore, we analysed the contribution of BK channels in bronchial tone and respiratory tract function using wild-tpye (wt) and BK channel-deficient (BK-/-) mice. The resting membrane potential of BK-/- bronchial smooth muscle cells was more depolarized when compared to wt, suggesting an increased open-probability of voltage-gated Ca2+ channels and subsequent increased bronchial tone. However, carbachol-induced broncho-constriction was reduced in vitro and in vivo. Furthermore, both isoprenalin-induced and cGMP-mediated bronchodilation were enhanced in BK-/- mice. In-depth analysis revealed a decreased expression of M3 receptors and an increased expression profile of NO/cGMP signaling proteins, whereas no alteration in the cAMP signaling pathway was observed. These results indicate that up-regulation of NO/cGMP signaling counterbalances for BK channel ablation, pointing to the predominant role of BK channel in regulation of bronchial tone. Thus, BK channel may serve as new drug target for the therapy of obstructive pulmonary diseases.