The human cytomegalovirus (HCMV) basic phosphoprotein pp150, encoded by the UL32 gene, together with the two other major phosphoproteins, pp65 (ppUL83) and pp71 (ppUL82) and several minor structural proteins, form the tegument around the viral nucleocapsid. Experiments were undertaken to locate the area of assembly of tegument proteins pp150 and pp65 and nucleocapsids in fibroblasts, in order to assess the functional role of these two structural proteins in HCMV morphogenesis. Whereas pp150 expression starts during the cytoplasmic maturation of HCMV, pp65 is expressed in the early and late phases of HCMV gene transcription. Western blot analysis of isolated cell fractions showed that pp150 is initially (48 h post-infection) localized in the nucleus, associated either with the nuclear membrane or with viral assembly regions, and later (72 h post-infection) in the cytoplasm. By indirect immunofluorescence, pp150 and pp65 could be detected in nuclear subcompartments and were strongly associated with the nuclear membrane. Using immunogold analysis by electron microscopy, pp65 was exclusively detected within the matrix of cytoplasmic and extracellular dense bodies and of dense body-like structures in the nucleoplasm. These were localized in close contact with hypertrophic nucleoli, in the proximity of developing nucleocapsids and in special patches at the inner nuclear membrane. Positive immunostaining of pp150 was observed at the surface of developing nucleocapsids concentrated within viral assembly regions in the nucleoplasm. Additionally, the tegument of cytoplasmic and extracellular virions was stained, whereas dense bodies or nuclear dense body-like structures did not react. Thus, the acquisition of the tegument protein pp150 seems to start in special nuclear subcompartments of the HCMV-infected fibroblasts.