The spontaneous interbilayer transfer of dehydroergosterol, a fluorescent cholesterol analog, was examined using small unilamellar phospholipid vesicles. The kinetic data were best fit by an equation of the form Aexp (-kt) + B. Qualitatively, the general trend of the half-time for transfer and the base values (B) obtained for dehydroergosterol resemble the corresponding values obtained in the earlier studies of cholesterol transfer. However, quantitative differences, which reflect the molecular structure of the sterol, were observed. Acrylamide quenching performed on the donor vesicles at different stages of the transfer indicated that a time-dependent organization of DHE within the vesicles occurs.