Acetylcholine (ACh) binding to atrial muscarinic receptors activates an inwardly rectifying K+ current (IK[ACh]) via a pertussis toxin-sensitive GTP-binding protein (GK). The muscarinic K+ channel (termed GIRK1) has been cloned, and the nucleotide sequence contains nine consensus sites for protein kinase C (PKC) phosphorylation (16). Dephosphorylation of the muscarinic K+ channel has been implicated in rapid IK[ACh] desensitization in the presence of agonist (13). Staurosporine is a widely used membrane-permeant inhibitor of PKC and other protein kinases (7), including G protein-coupled receptor kinases. We investigated the role of phosphorylation in the regulation of IK[ACh] by examining the effect of a variety of protein kinase inhibitors. Staurosporine produced a rapid and reversible dose-dependent decrease in IK[ACh], activated by either GTP or guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). Other PKC inhibitors, including calphostin C and K-252b, were without effect on GTP gamma S-activated IK[ACh]. In excised patches of atrial membrane under nonphosphorylating conditions (0 ATP, 1 mM 5'-adenylylimidodiphosphate), staurosporine reversibly reduced muscarinic K+ channel activity without altering single-channel current amplitude. These results suggest that staurosporine inhibits IK[ACh] by a mechanism independent of intracellular protein kinases.