Myristoylation of seven different a subunits of guanine nucleotide-binding regulatory proteins (G proteins) was examined by expressing these proteins in monkey kidney COS cells. Metabolic labeling studies of cells transfected with cytomegalovirus-based expression vectors indicated that[3H]myristate was incorporated into ail, a,2, a13, a0, at, and a, but not as subunits. The role of myristoylation in the association of a subunits with membranes was analyzed by sitedirected mutagenesis and by substitution of myristate with a less hydrophobic analog, 10-(propoxy)decanoate (li-oxamyristate). Myristoylation of a0 was blocked when an alanine residue was substituted for its amino-terminal glycine, as was association of the protein with membranes. Substitution of the myristoyl group with l1-oxamyristate affected the cellular distribution of a subset of acylated at subunits. The results are consistent with a model wherein the hydrophobic interaction of myristate with the bilayer permits continued association of the protein with the plasma membrane when G-protein a subunits dissociate from fty.Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) transduce signals across cell membranes by coupling receptors for chemical and sensory stimuli to effectors such as enzymes and ion channels. The best studied systems of this type are hormone-sensitive adenylyl cyclase (stimulated by the G protein GU) and the light-sensitive cyclic GMP phosphodiesterase in photoreceptor cells (stimulated by transducin or Gt) (1, 2). Other G proteins of relevance to this work include a group of four related substrates for pertussis toxin (Gil, GQ2, GU3, and GO), which appear to regulate ion channels, and GU, a newly appreciated G protein whose role in transmembrane signaling remains to be defined (3-5).G proteins are associated with the inner face of the plasma membrane, where they are positioned to interact with membrane-bound receptors and effectors. With the exception of Gt, detergent is required to extract G proteins from membranes. However, the deduced amino acid sequences of G protein a, f3, and y subunits lack obvious hydrophobic domains that could account for G protein-membrane interactions, and the molecular basis for the interaction of G proteins with the plasma membrane is largely unknown. Sternweis (6) demonstrated that the G-protein 13y subunit complex accounted for the detergent-binding properties of heterotrimeric G proteins. Furthermore, he demonstrated that ,fy could be incorporated into phospholipid vesicles and that G-protein a subunits would then interact stoichiometrically with fBy-containing vesicles.