alpha-Subunits of heterotrimeric G(i)-like proteins (alpha(i), alpha(o) and alpha(z)) associate with the cytoplasmic leaflet of the plasma membrane by means of N-terminally linked myristic acid and palmitic acid. An additional role for palmitate has been recently suggested by the observation that fusion with the palmitoylated N-terminus of alpha(i1) relocalizes cytosolic green-fluorescent-protein reporter to low buoyancy, Triton-insoluble membrane domains (TIFF; Triton-insoluble floating fraction), enriched with caveolin-1 [Galbiati, Volonté, Meani, Milligan, Lublin, Lisanti and Parenti (1999) J. Biol. Chem 274, 5843-5850]. Here we show that, upon transient expression in transfected COS-7 cells, myristoylated and palmitoylated alpha(o) (alpha(o)wt, where wt is wild-type) is exclusively found in TIFF, from where non-palmitoylated alpha(o)wt and alpha(o)C3S (Cys(3)-->Ser) mutant are excluded. Moreover, alpha(o) fused to N-terminally truncated human vasopressin V2 receptor (V2TR-alpha(o)), lacking myristate and palmitate, still localizes at the plasma membrane by means of first transmembrane helix of V2R, but is excluded from TIFF. Likewise, alpha(o)C3S does not partition into TIFF, even when its membrane avidity is enhanced by co-expression of betagamma-subunits. Thus membrane association, in the absence of added palmitate, is not sufficient to confer partitioning of alpha(o) within TIFF, suggesting that palmitoylation is a signal for membrane compartmentalization of dually acylated alpha-subunits.