The permeability of synthetic phosphatidylcholines has been studied with stopped-flow spectrometry by monitoring the initial shrinkage or swelling velocity of the sonicated vesicles. It was observed that aqueous diffusion was more rapid through dilinoleoyl- than through dioleoyl- which in turn was faster than through dipalmitoyl-phosphatidylcholine membranes. In contrast, when the vesicles were associated with poly-L-lysine, aqueous diffusion through dioleoyl- was greatly enhanced, whereas that through dilinoleoyl- and dipalmitoyl-phosphatidylcholine was diminished. When the vesicles were associated with polyglutamic acid, the permeability of dipalmitoyl decreased, that of dioleoyl increased, and there was little or no effect on dilinoleoyl vesicles. Measurements on the initial rate of optical density increase by dioleoyl- and dilinoleoyl-phosphatidylcholine vesicles, when they were mixed with polylysine solutions, showed that the rates of interaction by the two kinds of vesicles with the polypeptide were very different, dilinoleoyl reacting much more rapidly than dioleoyl. The observations indicate that phosphatidylcholine vesicles can interact with both acidic and basic polypeptides, and the reaction differs not only in relation to the charge on the polypeptide but also on the fatty acid composition of the lipid.