Effects of quinidine, disopyramide, and procainamide on the acetylcholine (ACh)-induced K+channel current were examined in single atrial cells, using the tight-seal, whole-cell clamp technique. The pipette solution contained guanosine-5'-triphosphate (GTP) or guanosine-5'-O-(3-thiotriphosphate) (GTP-yS, a nonhydrolysable GTP analogue). In GTP-loaded cells, not only ACh but also adenoslne induced a specific K+channel current via GTP-binding proteins (G) by activating muscarinic ACh or adenosine receptors. Quinidine and disopyramide depressed the ACh-induced K+current quite effectively. Procainamide had a weak inhibitory effect. Quinidine also depressed adenosine-induced K+current, while the effect of disopyramide on adenosine-induced current was much smaller than that on ACh-induced current. In GTP-γS-loaded cells, the K+channel was uncoupled from the receptors and was activated irreversibly, probably due to direct activation of G proteins by GTP-γS. Quinidine depressed the GTP-γS-induced K+current just as in the cases of ACh- and adenosine-induced currents of GTP-loaded cells. Disopyramide had only a weak inhibitory effect and procainamide showed no effect. From these results, it is strongly suggested that the major mechanisms underlying the anti-cholinergic effects of quinidine, disopyramide, and procainamide are different; quinidine may inhibit the muscarinic K+channel itself and/or G proteins, while disopyramide and high doses of procainamide may mainly block functions of muscarinic ACh receptors in atrial myocytes.