Phosphorylation of G protein-coupled receptors by beta-adrenergic receptor kinases (betaARK) requires the presence of G protein betagamma subunits. We have investigated the ability of the two betaARK isoforms to distinguish between defined recombinant betagamma subunits. betaARK2 had an about 25% lower specific activity than betaARK1 towards rhodopsin and the beta2-adrenergic receptor but the two kinases shared the selectivity for betagamma subunits: betagamma complexes consisting of beta1 or beta2 in combination with gamma2, gamma5, and gamma7 were more efficacious than those with gamma3 or beta1 gamma1. Thus, while betaARKs differentiate between defined betagamma subunits, betagamma complexes do not discriminate between betaARK isoforms.