Summary. This paper is a continuation of a series of physicochemical studies of vesicle-bound flavins. In this study a detailed analysis of the Sepharose 4B elution profile of various sonicated phospholipids is given which demonstrates the presence of three distinct particle populations. Electron microscopy reveals heterogeneous, multilamellar lipoid aggregates of a diameter of more than 500 ~ in fraction I, unilamellar, closed vesicles of 200 to 250 A diameter in fraction II, and ~previously unreported micellar structures of approximately 50 A diameter in fraction III. Fraction II represents vesicles very similar to those obtained by the "deoxycholate procedure." Fine structure analysis of corrected fluorescence and fluorescence polarization spectra of amphiphilic flavin (AFI 3) bound to vesicles prepared from various phospholipids demonstrates the specificity of the flavin/membrane interaction. When bound to fractions I, II and III of sonicated egg lecithin, AFI 3 exhibits different temperature (i.e. phase-) dependencies. This demonstrates that the flavin/membrane interaction depends strongly upon the particular lipid structure as well. Evidence is presented which defines the type of excited state of flavin involved in photochemical and photophysical reactions (triplet or singlet). Finally, the dependencies of the photoreactions of vesicle-associated flavins on parameters such as pH, temperature and ionic strength are discussed. Comparison of photoreactions of isotropic and anisotropic flavins also reveal clues for the various mechanisms involved.