Chemical evidence has been obtained for transbilayer movement of a di-walled and a tetra-walled molecular umbrella in large unilamellar vesicles (200 nm) derived from 1-palmitoyl-2-oleyol-sn-glycero-3-phoshatidylglycerol (POPG). A di-walled molecular umbrella (1), bearing 2-mercaptopyridine (2-MP) as a "reactive tag", was synthesized by reaction of N-1,N-3-spermidinebis[cholic acid amide] (3) with [N-1,2,3-benzotriazin-4(3H)one-yl]-3-(2-pyridyldithio)propionate [BPDP]. An analogous tetra-walled umbrella (2) was also prepared by condensing Fmoc-protected iminodiacetic acid with two molecules of 3, deprotecting the secondary amino group, and coupling the resulting intermediate (4) with BPDP. Reaction of vesicle-bound 1 with external glutathione (GSH) resulted in a rapid and quantitative release of 2-MP. A similar thiolate-disulfide interchange reaction that was carried out between membrane-bound I and GSH, which was captured within the aqueous interior of the vesicles, also resulted in rapid and complete release of 2-MP, These results, together with the fact that GSH does not permeate across the POPG vesicle membranes, provides compelling evidence for rapid transbilayer movement. Reaction of membrane-bound 2 with external GSH also resulted in the rapid and quantitative release of 2-MP. The significance of these findings, with regard to the current view of molecular size restrictions on membrane permeability, is briefly discussed.