Rotaviruses are non-enveloped, triple-shelled particles that cause enteritis in animals and humans. The interactions among the different viral proteins located in the three concentric layers make the rotavirus particle an excellent model for physico-chemical and biological studies of viral assemblage. SA11-4S rotaviruses subjected to high pressure were inactivated by more than five log units. After pressure treatment, the particles were recovered with slight structural changes when compared to the control. Electron microscopy suggested subtle changes in the viral outer layer in some pressurised particles. Fluorescence spectroscopy showed that much more dramatic changes were produced by urea denaturation than by pressure. Based on the fluorescence spectrum, the genome resistance to ribonuclease, and the absence of changes in hydrodynamic properties, there was little or no disruption of the capsid under pressure. On the other hand, hemagglutination assays indicated that the main component affected by pressure was the spike protein VP4, thus accounting for changes in interaction with host cells and greatly reduced infectivity. The changes leading to inactivation did not cause removal of VP4 from the outer capsid, as verified by size-exclusion chromatography. Antibodies raised against pressurised material were as effective as antibodies raised against the intact virus, based on their neutralisation titre in plaque reduction assays, enzyme-linked immunosorbent assays and direct interaction with the particle, as measured by gel-filtration chromatography. Therefore, the new conformation of the pressurised particle did not result in loss of immunogenicity. We propose that pressure alters the receptor-binding protein VP4 by triggering changes similar to those produced when the virus interacts with target cells. As the changes in VP4 conformation caused by pressure occur prior to virus exposure to target cells, it leads to non-infectious particles and may lead to the exposure of previously occult epitopes, important for vaccine development.