In plants one bona fide Galpha subunit has been identified, as well as a single Gbeta and two Ggamma subunits. To study the roles of lipidation motifs in the regulation of subcellular location and heterotrimer formation in living plant cells, GFP-tagged versions of the Arabidopsis thaliana heterotrimeric G protein subunits were constructed. Mutational analysis showed that the Arabidopsis Galpha subunit, GPalpha1, contains two lipidation motifs that were essential for plasma membrane localization. The Arabidopsis Gbeta subunit, AGbeta1, and the Ggamma subunit, AGG1, were dependent upon each other for tethering to the plasma membrane. The second Ggamma subunit, AGG2, did not require AGbeta1 for localization to the plasma membrane. Like AGG1, AGG2 contains two putative lipidation motifs, both of which were necessary for membrane localization. Interaction between the subunits was studied using fluorescence resonance energy transfer (FRET) imaging by means of fluorescence lifetime imaging microscopy (FLIM). The results suggest that AGbeta1 and AGG1 or AGbeta1 and AGG2 can form heterodimers independent of lipidation. In addition, FLIM-FRET revealed the existence of GPalpha1-AGbeta1-AGG1 heterotrimers at the plasma membrane. Importantly, rendering GPalpha1 constitutively active did not cause a FRET decrease in the heterotrimer, suggesting no dissociation upon GPalpha1 activation.