Production of an infectious herpes simplex virus (HSV) particle requires sequential progression of maturing virions through a series of complex assembly events. Capsids must be constructed in the nucleus, packaged with the viral genome, and transported to the nuclear periphery. They then bud into the nuclear membrane to acquire an envelope, traffic through the cytoplasm, and are released from the cell. Most of these phenomena are very poorly defined, and no suitable model system has previously been available to facilitate molecular analyses of genomic DNA packaging, capsid envelopment, and intracellular virion trafficking. We report the development of such an assay system for HSV type 1 (HSV-1). Using a reversible temperature-sensitive mutation in capsid assembly, we have developed conditions in which an accumulated population of immature capsids can be rapidly, efficiently, and synchronously chased to maturity. By assaying synchronized scaffold cleavage, DNA packaging, and acquisition of infectivity, we have demonstrated the kinetics with which these events occur. Kinetic and morphological features of intranuclear and extranuclear virion trafficking have similarly been examined by indirect immunofluorescence microscopy and electron microscopy. This system should prove a generally useful tool for the molecular dissection of many late events in HSV-1 biogenesis.