Abstract S-Bimanylthiocholesterol, a new fluorescent lipid, was synthesized from thiocholesterol and monobromobimane. Fluorescence excitation and emission spectra were measured in some organic solvents and after incorporation into unilamellar lipid vesicles. In the vesicles, excitation was at 389 nm and emission was at 459 nm. Very similar values were seen in methanol and in water containing 0.5% (v/v) Triton X-100. These spectral properties suggested that the bimane fluorophore was located in the lipid vesicles in a polar environment, close to the surface. The molar extinction coefficient at the excitation wavelength in methanol was 5400 M −1 · cm −1. Up to 7 mol% could be incorporated without self-quenching of the fluorescence. The exchange of S-bimanylthiocholesterol between vesicles was demonstrated, with an apparent first-order rate constant of 0.48 h −1. The extent of exchange at equilibrium indicated that lipid transfer was restricted to the probe molecules in the outer leaflets of the bilayers.