Following the infection of insect ovarian cells (Sf9) with recombinant bearing the cDNA coding for the rat muscarinic acetylcholine (ACh) receptor subtype m3, ionic flux across the membrane in response to the application of ACh was examined electrophysiologically. We show that ACh activates potassium currents. The response is abolished when cells are treated with pertussis toxin. No ACh-induced currents are observed from uninfected cells or cells infected with virus which do not contain the cDNA coding for ACh receptors in its genome. The characteristics of single channel currents show time-dependent changes following the application of ACh. Initially, ACh activates brief channel currents with a conductance of about 5 pS. The conductance level of channels gradually increases in steps to 10 pS and then to 20 pS and 40 pS. At the same time, channel open probability also increases. Thereafter, additional channels appear, opening and closing independently of, or at times in synchrony with, the original channel.