Capsid disassembly and genome release are critical steps in the lifecycle of a virus. However, their mechanisms are poorly understood, both in vivo and in vitro. Here, we have identified two in vitro disassembly pathways of the brome mosaic virus (BMV) by charge detection mass spectrometry and transmission electron microscopy. When subjected to a pH jump to a basic environment at low ionic strength, protein–RNA interactions are disrupted. Under these conditions, BMV appears to disassemble mainly through a global cleavage event into two main fragments: a near complete capsid that has released the RNA and the released RNA complexed to a small number of the capsid proteins. Upon slow buffer exchange to remove divalent cations at neutral pH, capsid protein interactions are disrupted. The BMV virions swell but there is no measurable loss of the RNA. Some of the virions break into small fragments, leading to an increase in the abundance of species with masses less than 1 MDa. The peak attributed to the BMV virion shifts to a higher mass with time. The mass increase is attributed to additional capsid proteins associating with the disrupted capsid protein–RNA complex, where the RNA is presumably partially exposed. It is likely that this pathway is more closely related to how the capsid disassembles in vivo, as it offers the advantage of protecting the RNA with the capsid protein until translation begins. - Copyright © 2020 American Chemical Society