Hepatitis B surface antigen (HBsAg) subvirus particles produced from yeast share immunological determinants with mature viruses, which enable the use of HBsAg as a potent antigen for human vaccination. Because the intimate structure of such pseudoviral particles is still a matter of debate, we investigated the robustness of the external barrier and its structure and dynamics using the noninvasive solid-state NMR technique. This barrier is made of 60% proteins and 40% lipids. Phospholipids represent 83% of all lipids, and chain unsaturation is of 72%. Dynamics was reported by embedding small amounts of deuterium chain-labeled unsaturated phospholipid into the external barrier of entire subviral particles, while controlling particle integrity by cryoelectron microscopy, tomography, and light scattering. Variable preparation modes were used, from mild incubation of small unilamellar vesicles to very stringent incorporation with freeze-drying. A lipid bilayer structure of 4- to 5-nm thickness was evidenced with a higher rigidity than that of synthetic phospholipid vesicles, but nonetheless reflecting a fluid membrane (50-52% of maximum rigidity) in agreement with the elevated unsaturation content. The HBsAg particles of 20- to 24-nm diameter were surprisingly found resistant to lyophilization, in such a way that trapped water inside particles could not be removed. These dual properties bring more insight into the mode of action of native subviral particles and their recombinant counterparts used in vaccines.-Grélard, A., Guichard, P., Bonnafous, P., Marco, S., Lambert, O., Manin, C., Ronzon, F., and Dufourc, E. J. Hepatitis B subvirus particles display both a fluid bilayer membrane and a strong resistance to freeze drying: a study by solid-state NMR, light scattering, and cryo-electron microscopy/tomography.