Signal transduction by G-protein coupled receptors (GPCRs) requires the physical interaction of receptors and heterotrimeric G-proteins and the subsequent conversion of G-proteins to the active state. GPCRs and G-proteins are thought to encounter each other by random collision after receptor activation, and active G-proteins are thought to dissociate into component Ga and Gbgsubunits. We have tested these ideas using FRAP to detect protein interactions in live cells. Experimental immobilization of one signaling molecule changes the lateral mobility of a second if the two interact stably. Immobilization of inactive GPCRs decreased the mobility of fluorescent protein (FP)-tagged Ga subunits, suggesting these molecules are associated prior to receptor activation. Immobilization of Ga subunits severely restricted the mobility of FP-tagged Gb[gamma], consistent with the known formation of stable Gab[gamma] heterotrimers. Constitutively active Gai3 Q204L subunits also decreased the mobility of FP-Gb[gamma], suggesting these proteins remain associated after activation. In contrast, constitutively active GaoA Q205L subunits were unable to decrease the mobility of FP-Gb[gamma], suggesting these proteins dissociate after activation. These results do not entirely conform to the standard model of GPCR signaling. Supported by a grant from the NIH.