Envelopment of herpes simplex virus type-1 (HSV-1) was investigated in relation to membrane differentiation in dissociated anterior pituitary cells. The number of cells stained positively with anti-HSV-1 serum was increased from 16 h to 31 h post infection. During this period, electron microscopy revealed that a number of nucleocapsids (unenveloped particles) were accumulated in the Golgi area, where they frequently became surrounded by a double membrane of short Golgi cisternae or by one with a Golgi associated endoplasmic reticulum lysosome (GERL)-like structure. The inner membrane of the cisterna surrounding the nucleocapsids showed regional specialization which was characterized by increased thickness and electron opacity. Acid phosphatase activity, a marker for GERL or trans Golgi cisternae, appeared in the cytoplasmic short cisternae surrounding the nucleocapsids, whereas glucose-6-phosphatase activity, a marker for the nuclear envelope or for endoplasmic reticulum, was not demonstrated in such cisternae. Monoclonal antibody against glycoprotein gD revealed that gD was localized in the trans Golgi membrane as well as in the envelope of the virion. The antibody-binding sites were highly concentrated in the area where Golgi membranes showed increased opacity. Furthermore, nucleocapsids were surrounded exclusively by gD-positive cisternal (Golgi or Golgi-derived) membranes. Thus, our results indicate that the envelope of HSV is derived from trans Golgi cisterna (GERL), and that some viral components, including gD, destined for the envelope may be assembled initially in the Golgi membrane, which is thereby transformed into the envelope of the virus.