In comparison with the α subunit of G proteins, the role of the β subunit in signaling is less well understood. During the regulation of effectors by the βγ complex, it is known that the β subunit contacts effectors directly, whereas the role of the β subunit is undefined in receptor-G protein interaction. Among the five G protein β subunits known, the β4 subunit type is the least studied. We compared the ability of βγ complexes containing β4 and the well characterized β1 to stimulate three different effectors: phospholipase C-β2, phospholipase C-β3, and adenylyl cyclase type II. β 4γ2 and β1γ2 activated all three of these effectors with equal efficacy. However, nucleotide exchange in a G protein constituting αoβ 4γ2 was stimulated significantly more by the M2 muscarinic receptor compared with αoβ1γ 2. Because αo forms heterotrimers with β 4γ2 and β1γ2 equally well, these results show that the β subunit type plays a direct role in the receptor activation of a G protein.