The presence of the Na-K-Cl cotransporter in airway smooth muscle was investigated by measuring 86Rb+ (as a marker for K+) and 36Cl- fluxes in the guinea pig trachealis. K+ uptake consisted of 1) a bumetanide- and furosemide-sensitive component, 2) a ouabain-sensitive component, and 3) a bumetanide- and ouabain-insensitive component. Bumetanide and furosemide inhibited K+ uptake with IC50s of 0.18 and 5.6 microM, respectively. Bumetanide-sensitive K+ uptake was reduced by the isosmotic replacement of extracellular Na+ and Cl- with choline and I-, respectively. Bumetanide caused a reduction in Cl- uptake, and the ratio of bumetanide-sensitive K+ to Cl-uptake was approximately 1:1.75. Bumetanide caused a decrease in 86Rb+ efflux, suggesting that the Na-K-Cl cotransporter mediates both K+ influx and efflux in airway smooth muscle. Ouabain caused an increase in bumetanide-sensitive 86Rb+ efflux from the guinea pig trachealis, which was prevented by exposure to a low-Na+ medium, suggesting that Na-K pump inhibition stimulates outward Na-K-Cl cotransport as a result of an increase in intracellular Na+ content.