The production and biochemical and physiocochemical analysis are described of recombinant-produced hepatitis B virus core antigen (HBcAg capsid) and the corresponding particle produced by a deletion mutant missing the C-terminal 39 residues (HBeAg). Conditions for producing HBeAg from HBcAg capsids by in vitro proteolysis are also described. The morphology and masses of these capsids were determined by scanning transmission electron microscopy. Both HBcAg and HBeAg capsids comprise two size classes that correspond to icosahedral lattices with triangulation numbers (T) of 3 and 4, containing 180 and 240 subunits per capsid, respectively. This dimorphism was confirmed by sedimentation equilibrium and sedimentation velocity measurements on a Beckman Optima XL-A analytical ultracentrifuge. More than 60% of HBcAg capsids were T = 4, whereas only 15-20% of HBeAg capsids were of this size class: the remainder, in each case, were T = 3. Circular dichroism and Raman spectroscopy were used to determine the overall secondary structures of HBcAg and HBeAg capsids. Both have high alpha-helical contents, implying that this capsid protein does not conform to the canonical beta-barrel motif seen for all plant and animal icosahedral viral capsids solved to date. We suggest that the C-terminal domain of HBcAg has a random coil conformation. In vitro dissociation of HBeAg capsids under relatively mild conditions yielded stable dimers. The reassociation of HBeAg dimers into capsids appears to be driven by hydrophobic processes at neutral pH. Capsid assembly is accompanied by little change in subunit conformation as judged by circular dichroism and fluorescence spectroscopy. The thermal stability of HBcAg capsids was compared calorimetrically with that of in vitro assembled HBeAg capsids. Both have melting temperatures > 90 degrees C, implying that the C-terminal region makes little difference to the thermal stability of HBcAg: nevertheless, we discuss its possible role in facilitating disassembly and the release of viral nucleic acid.