The molecular anatomy of the herpes simplex virus (HSV-1) capsid has been examined by conventional electron microscopy, cryoelectron microscopy combined with three-dimensional image reconstruction, and scanning transmission electron microscopy (STEM). Studies were carried out with purified capsids before and after treatment with urea and guanidine hydrochloride (GuHCl) at concentrations that maintain the capsid's icosahedral geometry, but selectively extract certain of its protein components. Treatment with 6.0 M urea was found to remove the pentons quantitatively from the capsid vertices, but it caused no appreciable loss of hexons. Penton loss was correlated with solubilization of a small amount of VP5, the major HSV-1 capsid protein, and the amount solubilized (6.1%) was in good agreement with the amount expected (6.3%) if pentons are each composed of five copies of VP5. We conclude that the pentons, like the hexons, are composed of VP5, which exists as a pentamer at the capsid vertices (the pentons) and as a hexamer in all other capsomers (the hexons). Control capsids and capsids extracted with 2.0 M GuHCl (G2.0 capsids) were examined by cryoelectron microscopy and the resulting images were employed to compute three-dimensional reconstructions. Also, the masses of control and G2.0 capsids were determined by dark-field STEM and the results were used to calculate copy numbers for the proteins present. The three-dimensional reconstructions showed that control and G2.0 capsids are similar in structure, except that G2.0 capsids lack all 12 pentons and 120 of the 320 trigonal nodules or "triplexes" that connect HSV-1 capsomers in groups of three. The missing triplexes are the ten closest to each capsid vertex. Thus, the tightness with which triplexes are bound to the VP5 matrix varies according to position on the T = 16 icosahedral surface lattice, those closest to the pentons being most easily detached. Biochemical analyses revealed partial loss of the minor capsid proteins VP19 and VP23 in G2.0 compared to control capsids. Taking into account the STEM data on capsid protein stoichiometry, we propose that HSV-1 triplexes are heterotrimers composed of one copy of VP19 and two copies of VP23.