The activated heterotrimeric guanine nucleotide binding (G) protein Gk, at subpicomolar concentrations, mimics muscarinic stimulation of a specific atrial potassium current. Reconstitution studies have implicated the alpha and beta gamma subunits as mediators, but subunit coupling by the endogenous G protein has not been analyzed. To study this process, a monoclonal antibody (4A) that binds to alpha k but not to beta gamma was applied to the solution bathing an inside-out patch of atrial membrane; the antibody blocked carbachol-activated currents irreversibly. The state of the endogenous Gk determined its susceptibility to block by the antibody. When agonist was absent or when activation by muscarinic stimulation was interrupted by withdrawal of guanosine triphosphate (GTP) in the presence or absence of guanosine diphosphate (GDP), the effects of the antibody did not persist. Thus, monoclonal antibody 4A blocked muscarinic activation of potassium channels by binding to the activated G protein in its holomeric form or by binding to the dissociated alpha subunit.