Several classes of voltage-gated Ca²⁺ channels (VGCCs) are inhibited by G proteins activated by receptors for neurotransmitters and neuromodulatory peptides. Evidence has accumulated to indicate that for non-L-type Ca²⁺ channels the executing arm of the activated G protein is its βγ dimer (Gβγ). We report below the existence of two Gβγ-binding sites on the A-, B-, and E-type α₁ subunits that form non-L-type Ca²⁺ channels. One, reported previously, is in loop 1 connecting transmembrane domains I and II. The second is located approximately in the middle of the ca. 600-aa-long C-terminal tails. Both Gβγ-binding regions also bind the Ca²⁺ channel β subunit (CCβ), which, when overexpressed, interferes with inhibition by activated G proteins. Replacement in α_1E of loop 1 with that of the G protein-insensitive and Gβγ-binding-negative loop 1 of α_1C did not abolish inhibition by G proteins, but the exchange of the α_1E C terminus with that of α_1C did. This and properties of α_1E C-terminal truncations indicated that the Gβγ-binding site mediating the inhibition of Ca²⁺ channel activity is the one in the C terminus. Binding of Gβγ to this site was inhibited by an α₁-binding domain of CCβ, thus providing an explanation for the functional antagonism existing between CCβ and G protein inhibition. The data do not support proposals that Gβγ inhibits α₁ function by interacting with the site located in the loop I–II linker. These results define the molecular mechanism by which presynaptic G protein-coupled receptors inhibit neurotransmission.