Application of a muscarinic agonist to the extracellular surface of membrane patches from airway smooth muscle cells resulted in an inhibition of calcium-activated potassium (KCa) channels in outside-out patches. Methacholine (50 microM) inhibited channel activity at physiological cytosolic calcium concentrations and resulted in a marked shift in channel open-time kinetics. In inside-out patches, KCa channels were inhibited upon addition of GTP (100 microM) when methacholine was present in the patch pipette. Muscarinic inhibition was blocked when guanosine 5'-O-(2-thiodiphosphate) was used to compete with endogenous GTP in outside-out or inside-out experiments. Pretreatment of dissociated cells with pertussis toxin (0.1 micrograms/ml) blocked muscarinic inhibition of the channel in a time-dependent fashion. These results demonstrate, at the single-channel level, a coupling between muscarinic receptor stimulation and inhibition of KCa in smooth muscle and demonstrate the guanine nucleotide dependence of this coupling.