The gamma-subunits of heterotrimeric guanine nucleotide-binding regulatory proteins (G-proteins) are isoprenylated and alpha-carboxyl methylated at their COOH-terminal cysteine residues. These modifications are necessary for membrane attachment of the beta gamma complex, but a requirement of an additional factor has been proposed for the stable binding. We explored a possible contribution of the blocked amino terminus of beta-subunits of bovine photoreceptor G-protein, transducin (T alpha/T beta gamma = Gt alpha/beta 1 gamma 1), and of three beta gamma complexes (beta 1 gamma 2, beta 1 gamma 3, and beta 1 gamma 7) purified from bovine brains. Structural analyses revealed that every beta 1-subunit has an N-acetylated serine, which is unlikely to contribute to the membrane association. Since neither protease nor heat treatment of photoreceptor membranes affected the membrane binding of T beta gamma, it seems unlikely that rhodopsin (or other membrane proteins) serves as an anchor protein for accepting T beta gamma. In fact, T beta gamma bound to phospholipid large unilamellar vesicles (LUVs), of which the polar head groups strongly influenced the binding: T beta gamma alone showed 2-fold higher binding for negatively charged phosphatidylserine-LUVs than for neutral phosphatidylcholine (PC)-LUVs, while the affinity of T alpha/T beta gamma complex for the phosphatidylserine-LUVs was lower than that for the PC-LUVs. These results indicate that 1) an ionic interaction between T beta gamma and membrane surface plays an important role in the stable membrane association, and 2) the domain(s) of T beta gamma responsible for the association would be different between trimeric and dissociated states. We also found that synthetic peptides corresponding to the COOH-terminal region of T gamma inhibited T alpha-T beta gamma interaction only when the peptides were isoprenylated. This suggests that the isoprenyl moiety is located at the contact site between the subunits, not at the membrane-binding domain, when T beta gamma is complexed with T alpha.