Hypothesis: Surfactant vesicles composed of ditallowethylesterdimethylammonium chloride (DEEDMAC), a cationic double tail surfactant, are commonly present in personal care industrial formulations such as fabric softeners. There is significant interest in formulating vesicle dispersions, investigation of stability, characterization of their structure and flow properties due to the biodegradable nature of DEEDMAC. Experiments: We investigate the formation and structure of unilamellar nanovesicles having a shell made of DEEDMAC and a core containing water. We use bright field optical microscopy to elucidate the formation mechanism, and a combination of small angle neutron scattering (SANS), cryogenic transmission electron microscopy (cryo-TEM), viscometry, densitometry, dynamic light scattering (DLS), small angle X-ray scattering (SAXS) and zeta potential measurements to determine the nanostructure of well-defined surfactant nanovesicles (similar to 15 nm diameter). Findings: We report methods for the determination of volume fraction of nanovesicles and vesicle density, which are crucial for quantitative estimation of nanovesicle performance in practical applications and for predicting vesicle stability. The nanovesicle volume fraction can be obtained directly from the intrinsic viscosity and density. The robust method presented here is simple and effective as confirmed by quantitative agreement of the results with independent SANS measurements. (C) 2014 Elsevier Inc. All rights reserved.