Electron cryo-microscopy has become a routine technique to determine the structure of biochemically purified herpes simplex virus capsid particles. This chapter describes the procedures of specimen preparation by cryopreservation; low dose and low temperature imaging in an electron cryo-microscope; and data processing for reconstruction. This methodology has yielded subnanometer resolution structures of the icosahedral capsid shell where α-helices and β-sheets of individual subunits can be recognized. A relaxation of the symmetry in the reconstruction steps allows us to resolve the DNA packaging protein located at one of the 12 vertices in the capsid.