The unusual spontaneous formation of submicrometer-sized vesicles from a small, nonamphiphilic bis-biuret difluorene derivative upon dissolution of the solid in an anhydrous organic solvent was investigated using multiple scattering techniques. Time-resolved light scattering (TLS) measurements confirm that the self-assembly process is driven by hydrogen-bonding interactions, leading to the formation of vesicles at a critical concentration similar to 1 x 10(-4) M in tetrahydrofuran as determined by absorbance and surface tension measurements. Results from cryogenic-scanning electron microscopy (cryo-SEM), dynamic light scattering (DLS), and small-angle X-ray scattering (SAXS) experiments are consistent with the existence of vesicle-like aggregates in solution. DLS studies indicate a broad distribution of aggregates with a mean hydrodynamic radius < R-H > = 303 nm (polydispersity =0.49). SAXS profiles show two decay regimes (low-Q decay, very large aggregates; large-Q decay, smaller species). The analysis models the large aggregates as vesicles (hollow spheres) with a mean external radius R-o = 750 nm and an internal radius R-i = 720 nm while the smaller aggregates have a mean radius R = 2.2 nm. The results obtained by cryo-SEM show spherical aggregates of vesicles size in the range ca. 100 nm to 1 mu m. Transmission electron microscopy (TEM) micrographs evidence the presence of aggregates whose morphology is compatible with budding and pearling processes as possible mechanisms for the formation of vesicles.