During hepatitis B virus (HBV) infections, subviral particles (SVP) consisting only of viral envelope proteins and lipids are secreted. Heterologous expression of the small envelope protein S in mammalian cells is sufficient for SVP generation. S is synthesized as a transmembrane protein with N-terminal (TM1), central (TM2), and hydrophobic C-terminal (HCR) transmembrane domains. The loops between TM1 and TM2 (the cytosolic loop [CL]) and between TM2 and the HCR (the luminal loop [LL]) are located in the cytosol and the endoplasmic reticulum (ER) lumen, respectively. To define the domains of S mediating oligomerization during SVP morphogenesis, S mutants were characterized by expression in transiently transfected cells. Mutation of 12 out of 15 amino acids of TM1 to alanines, as well as the deletion of HCR, still allowed SVP formation, demonstrating that these two domains are not essential for contacts between S proteins. Furthermore, the oligomerization of S was measured with a fluorescence-activated cell sorter (FACS)-based Förster resonance energy transfer (FRET) assay. This approach demonstrated that the CL, TM2, and the LL independently contributed to S oligomerization, while TM1 and the HCR played minor roles. Apparently, intermolecular homo-oligomerization of the CL, TM2, and the LL drives S protein aggregation. Detailed analyses revealed that the point mutation C65S in the CL, the mutation of 13 out of 19 amino acids of TM2 to alanine residues, and the simultaneous replacement of all 8 cysteine residues in the LL by serine residues blocked the abilities of these domains to support S protein interactions. Altogether, specific domains and residues in the HBV S protein that are required for oligomerization and SVP generation were defined. The small hepatitis B virus envelope protein S has the intrinsic ability to direct the morphogenesis of spherical 20-nm subviral lipoprotein particles. Such particles expressed in yeast or mammalian cells represent the antigenic component of current hepatitis B vaccines. Our knowledge about the steps leading from the initial, monomeric, transmembrane translation product of S to SVP is very limited, as is our information on the structure of the complex main epitope of SVP that induces the formation of protective antibodies after vaccination. This study contributes to our understanding of the oligomerization process of S chains during SVP formation and shows that the cytoplasmic loop, one membrane-embedded domain, and the luminal loop of S independently drive S-S oligomerization.