Enveloped viruses of eukaryotes obtain their membrane by budding through a cellular membrane. Therefore, most frequently the lipid composition of the virion envelope reflects that of the membrane where budding took place. In the case of herpes simplex viruses, nucleocapsids assemble in the nucleus and bud through the inner nuclear membrane. The pathway from the perinuclear space to the extracellular medium is as yet poorly understood. Here we demonstrate that the phospholipid composition of extracellular herpes simplex virions differs from that of nuclei isolated from the infected cells. The viral membrane contains threefold higher concentrations of sphingomyelin and phosphatidylserine. These lipids are typically enriched in the Golgi apparatus and plasma membrane. The data are in agreement with a model in which herpes simplex virus, after budding through the inner nuclear membrane, loses its envelope by fusing with the outer nuclear membrane and obtains a new membrane by budding into a compartment late in the exocytotic pathway, very likely the Golgi apparatus or membranes derived from it. Alternatively, because the perinuclear space is continuous with the ER lumen, the virus after its first budding may be transported through the exocytotic pathway without ever leaving the lumen of the subsequent compartments. In that case, either the virions, while budding through the nuclear membrane select for sphingomyelin and phosphatidylserine, or the original lipids of the viral envelope are exchanged for lipids of an exocytotic membrane, most likely by a transient membrane continuity between the virion and the vesicle by which it is surrounded. Light particles, virus-like particles that lack capsid and DNA but contain tegument and envelope proteins, displayed the same lipid composition as complete herpes simplex virions, suggesting that they also acquired their envelope from a Golgi membrane.