The assembly of human cytomegalovirus (HCMV) is thought to be similar to that which has been proposed for alphaherpesviruses and involve envelopment of tegumented subviral particles at the nuclear membrane followed by export from the cell by a poorly defined pathway. However, several studies have shown that at least two tegument virion proteins remain in the cytoplasm during the HCMV replicative cycle, thereby suggesting that HCMV cannot acquire its final envelope at the nuclear envelope. We investigated the assembly of HCMV by determining the intracellular trafficking of the abundant tegument protein pp150 (UL32) in productively infected human fibroblasts. Our results indicated that pp150 remained within the cytoplasm throughout the replicative cycle of HCMV and accumulated in a stable, juxtanuclear structure late in infection. Image analysis using a variety of cell protein-specific antibodies indicated that the pp150-containing structure was not a component of the endoplasmic reticulum, (ER), ER-Golgi intermediate compartment, cis or medial Golgi, or lysosomes. Partial colocalization of the structure was noted with the trans-Golgi network, and it appeared to lie in close proximity to the microtubule organizing center. Two additional tegument proteins (pp28 and pp65) and three envelope glycoproteins (gB, gH, and gp65) localized in this same structure late infection. This compartment appeared to be relatively stable since pp150, pp65, and the processed form of gB could be coisolated following cell fractionation. Our findings indicated that pp150 was expressed exclusively within the cytoplasm throughout the infectious cycle of HCMV and that the accumulation of the pp150 in this cytoplasmic structure was accompanied by at least five other virion proteins. These results suggested the possibility that this virus-induced structure represented a cytoplasmic site of virus assembly.