Egress of herpes capsids from the nucleus to the plasma membrane is a complex multistep transport event that is poorly understood. The current model proposes an initial envelopment at the inner nuclear membrane of capsids newly assembled in the nucleus. The capsids are then released in cytosol by fusion with the outer nuclear membrane. They are finally reenveloped at a downstream organelle before traveling to the plasma membrane for their extracellular release. Although the trans-Golgi network (TGN) is often cited as a potential site of reenvelopment, other organelles have also been proposed, including the Golgi, endoplasmic reticulumGolgi intermediate compartment, aggresomes, tegusomes, and early or late endosomes. To clarify this important issue, we followed herpes simplex virus type 1 egress by immunofluorescence under conditions that slowed intracellular transport and promoted the accumulation of the otherwise transient reenvelopment intermediate. The data show that the capsids transit by the TGN and point to this compartment as the main reenvelopment site, although a contribution by endosomes cannot formally be excluded. Given that viral glycoproteins are expected to accumulate where capsids acquire their envelope, we examined this prediction and found that all tested could indeed be detected at the TGN. Moreover, this accumulation occurred independently of capsid egress. Surprisingly, capsids were often found immediately adjacent to the viral glycoproteins at the TGN.The release of newly assembled herpesviruses requires passage through several host membranes by mechanisms that are poorly understood. Following their assembly and maturation in the nucleus, the capsids acquire a primary envelope by budding through the inner nuclear membrane (16,58,82) to end up in the perinuclear space, which is contiguous with the endoplasmic reticulum (ER) lumen. One model suggests these perinuclear virions escape the cell via the host biosynthetic pathway, which requires an obligatory transit through the Golgi (16, 44). However, the currently favored model proposes that the enveloped perinuclear capsids fuse with the outer nuclear membrane to produce naked cytosolic capsids (81,82). These would in turn acquire a secondary envelope downstream from an intracellular compartment, before reaching the plasma membrane and being released extracellularly by a second fusion event. This reenvelopment model appears valid for several, if not all, members of the herpesvirus family and is supported by several approaches, including electron microscopy (EM), immunofluorescence, freeze fracture, lipid content, as well as analysis of the site of tegument addition and the use of various viral mutants (23,53,54).Herpes simplex virus type 1 (HSV-1) is a member of the herpes family that has extensively been studied for egress. Unfortunately, its relatively short life cycle makes it difficult to analyze the vectorial movement of the virus during its rapid egress. Furthermore, EM analysis often gives a static snapshot without detailed information ...