A fluorescence-enhancement assay has been used to monitor the association of a series of fluorescent isoprenylated di- to tetrapeptides, whose sequences represent the carboxyl termini of several isoprenylated proteins (Ki-ras, ral 1, rac 2, and rho C), with phospholipid vesicles. These lipopeptides, containing mainly hydrophilic amino acid residues, all rapidly equilibrate (in seconds or faster) between the aqueous phase and the outer surfaces of lipid vesicles, in a manner that is well-modeled as a simple two-phase partitioning equilibrium. Farnesylated or geranylgeranylated peptides with methylated C-terminal cysteine residues exhibit half-maximal binding to 9:1 phosphatidylcholine (PC)/phosphatidylethanolamine (PE) vesicles at lipid concentrations on the order of 5-40 microM or 200-800 nM, respectively. Removal of the methyl group from the carboxyl-terminal cysteine residue decreases the affinity of a given lipopeptide for neutral (PC/PE) vesicles by 10- to 20-fold and the affinity for vesicles with a physiological surface charge by 40-fold or more. Cysteine-linked farnesyl and geranylgeranyl residues are found to be equivalent to cysteine-linked n-alkyl chains of roughly 11 and 14 carbons, respectively, in the strength of their interactions with lipid bilayers. Variations in vesicle lipid composition (cholesterol or aminophospholipid content) only modestly alter the affinity of isoprenylated peptides for the lipid bilayer. Our data suggest that a C-terminal geranylgeranylcysteine or O-methylated farnesylcysteine residue can by itself confer efficient (but rapidly reversible) membrane binding to proteins bearing these modifications, while an unmethylated C-terminal farnesylcysteine residue by itself would be only marginally efficient as a membrane 'anchor' under physiological conditions.