Heterotrimeric G proteins mediate physiological processes ranging from phototransduction to cell migration. In the accepted model of G protein signaling, Gαβγ heterotrimers physically dissociate after activation, liberating free Gα subunits and Gβγ dimers. This model is supported by evidence obtained in vitro with purified proteins, but its relevance in vivo has been questioned. Here, we show that at least some heterotrimeric G protein isoforms physically dissociate after activation in living cells. Gα subunits extended with a transmembrane (TM) domain and cyan fluorescent protein (CFP) were immobilized in the plasma membrane by biotinylation and cross-linking with avidin. Immobile CFP-TM-Gα greatly decreased the lateral mobility of intracellular Gβ1γ2-YFP, indicating the formation of stable heterotrimers. A GTPase-deficient (constitutively active) mutant of CFP-TM-GαoA lost the ability to restrict Gβ1γ2-YFP mobility, whereas GTPase-deficient mutants of CFP-TM-Gαi3 and CFP-TM-Gαs retained this ability. Activation of cognate G protein-coupled receptors partially relieved the constraint on Gβ1γ2-YFP mobility induced by immobile CFP-TM-GαoA and CFP-TM-Gαi3 but had no effect on the constraint induced by CFP-TM-Gαs. These results demonstrate the physical dissociation of heterotrimers containing GαoA and Gαi3 subunits in living cells, supporting the subunit dissociation model of G protein signaling for these subunits. However, these results are also consistent with the suggestion that G protein heterotrimers (e.g., Gαs) may signal without physically dissociating.