Select lipid-anchored proteins such as glycosylphosphatidylinositol (GPI)-anchored proteins and nonreceptor tyrosine kinases may preferentially partition into sphingomyelin-rich and cholesterol-rich plasmalemmal microdomains, thereby acquiring resistance to detergent extraction. Two such domains, caveolae and lipid rafts, are morphologically and biochemically distinct, contain many signaling molecules, and may function in compartmentalizing cell surface signaling. Subfractionation and confocal immunofluorescence microscopy reveal that, in lung tissue and in cultured endothelial and epithelial cells, heterotrimeric G proteins (G_i, G_q, G_s, and G_{β γ}) target discrete cell surface microdomains. G_q specifically concentrates in caveolae, whereas G_i and G_s concentrate much more in lipid rafts marked by GPI-anchored proteins (5′ nucleotidase and folate receptor). G_q, apparently without G_{β γ} subunits, stably associates with plasmalemmal and cytosolic caveolin. G_i and G_sinteract with G_{β γ} subunits but not caveolin. G_i and G_s, unlike G_q, readily move out of caveolae. Thus, caveolin may function as a scaffold to trap, concentrate, and stabilize G_q preferentially within caveolae over lipid rafts. In N2a cells lacking caveolae and caveolin, G_q, G_i, and G_s all concentrate in lipid rafts as a complex with G_{β γ}. Without effective physiological interaction with caveolin, G proteins tend by default to segregate in lipid rafts. The ramifications of the segregated microdomain distribution and the G_q-caveolin complex without G_{β γ} for trafficking, signaling, and mechanotransduction are discussed.