Stimulated muscarinic acetylcholine receptors (M2Rs) release Gβγ subunits, which slow heart rate by activating a G protein-gated K+ channel (GIRK). Stimulated β2 adrenergic receptors (β2ARs) also release Gβγ subunits, but GIRK is not activated. This study addresses the mechanism underlying this specificity of GIRK activation by M2Rs. K+ currents and bioluminescence resonance energy transfer between labelled G proteins and GIRK show that M2Rs catalyze Gβγ subunit release at higher rates than β2ARs, generating higher Gβγ concentrations that activate GIRK and regulate other targets of Gβγ. The higher rate of Gβγ release is attributable to a faster G protein coupled receptor - G protein trimer association rate in M2R compared to β2AR. Thus, a rate difference in a single kinetic step accounts for specificity.,© 2018, Touhara and MacKinnon.