P/Q Ca(2+) channel activity is inhibited by G protein-coupled receptor activation. Channel inhibition requires a direct Gbetagamma binding onto the pore-forming subunit, Ca(v)2.1. It is characterized by biophysical changes, including current amplitude reduction, activation kinetic slowing, and an I-V curve shift, which leads to a reluctant mode. Here, we have characterized the contribution of the auxiliary beta(3)-subunit to channel regulation by G proteins. The shift in I-V to a P/Q reluctant mode is exclusively observed in the presence of beta(3). Along with the observation that Gbetagamma has no effect on the I-V curve of Ca(v)2.1 alone, we propose that the reluctant mode promoted by Gbetagamma corresponds to a state in which the beta(3)-subunit has been displaced from its channel-binding site. We validate this hypothesis with a beta(3)-I-II(2.1) loop chimera construct. Gbetagamma binding onto the I-II(2.1) loop portion of the chimera releases the beta(3)-binding domain and makes it available for binding onto the I-II loop of Ca(v)1.2, a G protein-insensitive channel. This finding is extended to the full-length Ca(v)2.1 channel by using fluorescence resonance energy transfer. Gbetagamma injection into Xenopus oocytes displaces a Cy3-labeled beta(3)-subunit from a GFP-tagged Ca(v)2.1 channel. We conclude that beta-subunit dissociation from the channel complex constitutes a key step in P/Q calcium channel regulation by G proteins that underlies the reluctant state and is an important process for modulating neurotransmission through G protein-coupled receptors.