Unilamellar vesicles of anionic, phosphate-functionalized lipid 1 (DPGPNP, 1,2-di-O-hexadecanoyl-rac-glyceryl p-nitrophenyl phosphate, sodium salt) have been prepared both by sonication and by extrusion. The phosphate head groups are hydrolytically stable at pH 7 but can be specifically cleaved, at 25 degrees C, on the exo surface by adding, to preformed aggregates, lanthanide(III) cations which are unable to permeate across the bilayer. The exovesicular specificity disappears when the cleavage is performed above the phase transition temperature of the vesicles not because of facilitated permeation of the cations but because of the increased rate of flip-flop of the lipid. Addition of lipophilic amines (N-hexadecyl-N,N',N'-trimethylethylenediamine, TMED C-16; N-hexadecylhistamine, C-16-His, hexadecylamine, C16NH2) and, to a lesser extent, the cationic surfactant cetyltrimethylammonium bromide, CTABr, induces the cleavage of the remaining fraction of lipid in exo-cleaved vesicles. The additives affect the permeability of the bilayer, likely creating "leaky patches" in the vesicular membrane and, in the case of the amines, also transporting the cations across the bilayer by forming lipophilic complexes.