The short-term desensitization of the acetylcholine (ACh)-induced K+ channel current was examined in single atrial cells of guinea-pig heart. The tight-seal whole cell voltage clamp technique was used. The solution in the pipettes contained GTP or guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma S, a non-hydrolyzable GTP analogue). In GTP-loaded cells, ACh evoked a specific K+ channel current via GTP-binding proteins (G) in a dose-dependent manner. The K+ current showed agonist-dependent desensitization similar to those reported in other cardiac tissues (Nilius 1983; Carmeliet and Mubagwa 1986). The cellular response to ACh was also desensitized by activation of P1-purinergic receptors with adenosine (Ado). In GTP-gamma S-loaded cells, the K+ current was gradually induced even in the absence of agonists, probably due to direct activation of G proteins by GTP-gamma S. In the early phase of the spontaneous current increase, ACh evoked a large current transiently. As the GTP-gamma S-induced activation of the current progressed, the magnitude of the ACh-evoked current transient became smaller and finally negligible. Similar results were obtained when Ado was used as an agonist instead of ACh to induce the K+ current. Therefore, it is indicated that the agonist-receptor interaction may not be essential for the desensitization of ACh-induced K+ current in atrial myocytes.