Previously, the livers of patients suffering from acute liver failure (ALF), a potentially fatal syndrome arising from infection by Hepatitis B Virus (HBV), were found to contain massive amounts of an antibody specific for the core antigen (HBcAg) capsid. We have used cryo-electron microscopy and molecular modeling to define its epitope. HBV capsids are icosahedral shells with 25 angstrom-long dimeric spikes, each a 4-helix bundle, protruding from the contiguous "floor". Of the anti-HBcAg antibodies previously characterized, most bind around the spike tip while one binds to the floor. The ALF-associated antibody binds tangentially to a novel site on the side of the spike. This epitope is conformational. The Fab binds with high affinity to its principal determinants but has lower affinities for quasi-equivalent variants. The highest occupancy site is on one side of a spike, with no detectable binding to the corresponding site on the other side. Binding of one Fab per dimer was also observed by analytical ultracentrifugation. The Fab did not bind to the e-antigen dimer, a non-assembling variant of capsid protein. These findings support the propositions that antibodies with particular specificities may correlate with different clinical expressions of HBV infection and that antibodies directed to particular HBcAg epitopes may be involved in ALF pathogenesis. Published by Elsevier Inc.