Clinically, nitric oxide (NO⋅) is widely used as a pulmonary vaso- and bronchodilator agent. However, the precise molecular mechanisms by which NO⋅ induces smooth muscle relaxation are not well established. It has been suggested that NO⋅ relaxes airway smooth muscle (ASM) via a 3′,5′-cyclic guanosine monophosphate (cGMP)-dependent pathway, and our previous work has shown that Ca²⁺-activated K⁺ (K_Ca) channels are susceptible to cGMP-dependent protein kinase (PKG)-dependent phosphorylation (A. Alioua, J. P. Huggins, and E. Rousseau. Am. J. Physiol. 1995;268:L1057–L1063). To assess whether K_Ca channels are also directly activated by NO⋅ or one of its derivatives such as peroxynitrite, the activity of these channels was measured upon fusion of sarcolemmal vesicles derived from bovine tracheal smooth muscle cells into planar lipid bilayers (PLB). It was found that in the absence of adenosine triphosphate (ATP), cGMP, and cGMP-dependent protein kinase, NO⋅ donors such as 1-propanamine-3-(2-hydroxy-2-nitroso-1-propylhydrazine) (PAPA NONOate) or 3-morpholinosydnonimine hydrochloride (SIN-1) in the presence of superoxide dismutase (SOD), added on either side of the bilayer, caused a concentration- dependent increase in the open probability (Po) of K_Ca channels without altering their unitary conductance. Release of NO⋅, which was measured by chemiluminescence analysis in parallel experiments, affected the gating behavior of K_Ca channels in the presence of SOD and ethyleneglycol-bis-(β-aminoethyl ether)- N,N′-tetraacetic acid (EGTA) by reducing the mean closed times and increasing the number and duration of short open events. PAPA NONOate, a true NO⋅ donor, had similar effects in the presence of ethylenediaminetetraacetic acid (EDTA), a heavy-metal chelator, and K-urate, a peroxynitrite scavenger. Addition of either 5 mM dithiothreitol (DTT) or 5 mM reduced glutathione (GSH), as well as 5 mM N-ethylmaleimide (NEM)—an alkylating agent—to the trans (intracellular) side of an experimental chamber slightly increased channel Po but prevented further channel activation by NO⋅ donors. However, neither DTT nor GSH was able to reverse the effect of NO⋅. In contrast to SIN-1, DTT had no effect when added to the cis (extracellular) side of the chamber. This suggests that the effect of NO⋅ is most likely due to a chemical modification (nitrothiosylation) of intracellular sulfhydryl group(s). Neither PAPA NONOate (NO⋅), nor SIN-1 had any effect on sarcolemmal Cl⁻ channels reconstituted from the same membrane preparations. Pharmacomechanical measurements made on epithelium-denuded rat bronchus showed that 100 nM charybdotoxin decreased the sensitivity of bronchial smooth muscle to SIN-1-induced relaxations. Altogether, our data suggest that NO-induced bronchorelaxation occurs partly via a direct activation of K_Ca channels, possibly through a covalent interaction with the cytoplasmic side of their α subunit.