The muscarinic potassium current IK(ACh) of atrial myocytes can be evoked in the absence of agonists by intracellular application of stable GTP analogs (GXP). This receptor-independent opening of K(ACh) channels is a consequence of the direct activation of the guanyl nucleotide binding protein Gk that couples muscarinic receptors to K(ACh) channels, and was previously thought to be unaffected by subsequent application of agonist. We report here that in the presence of GTP, application of a pulse of muscarinic agonist to atrial cells can abolish the GXP-induced IK(ACh). The results imply that in intact cells the agonist-bound receptor can interact with Gk not only in its inactive, GDP-bound form, but also in its active, GXP-bound form in a process that promotes the release of guanine nucleotide from its binding site.