Human cytomegalovirus (HCMV) replicates in the nuclei of infected cells. Successful replication therefore depends on particle movements between the cell cortex and nucleus during entry and egress. To visualize HCMV particles in living cells, we have generated a recombinant HCMV expressing enhanced green fluorescent protein (EGFP) fused to the C terminus of the capsid-associated tegument protein pUL32 (pp150). The resulting UL32-EGFP-HCMV was analyzed by immunofluorescence, electron microscopy, immunoblotting, confocal microscopy, and time-lapse microscopy to evaluate the growth properties of this virus and the dynamics of particle movements. UL32-EGFP-HCMV replicated similarly to wild-type virus in fibroblast cultures. Green fluorescent virus particles were released from infected cells. The fluorescence stayed associated with particles during viral entry, and fluorescent progeny particles appeared in the nucleus at 44 h after infection. Surprisingly, strict colocalization of pUL32 and the major capsid protein pUL86 within nuclear inclusions indicated that incorporation of pUL32 into nascent HCMV particles occurred simultaneously with or immediately after assembly of the capsid. A slow transport of nuclear particles towards the nuclear margin was demonstrated. Within the cytoplasm, most particles performed irregular short-distance movements, while a smaller fraction of particles performed centripetal and centrifugal long-distance movements. Although numerous particles accumulated in the cytoplasm, release of particles from infected cells was a rare event, consistent with a release rate of about 1 infectious unit per h per cell in HCMV-infected fibroblasts as calculated from single-step growth curves. UL32-EGFP-HCMV will be useful for further investigations into the entry, maturation, and release of this virus.Like all herpesviruses, human cytomegalovirus (HCMV) replicates in the nucleus of the infected cell (18). This aspect of the herpesviral replication strategy entails the requirement for various particle movements during the replicative cycle, in particular translocation of penetrated virus particles from the cell cortex towards the nucleus, egress of newly synthesized virus particles out of the nucleus, and translocation of tegumented and enveloped virus progeny towards the periphery of the cell in order to release infectious virus to surrounding cells (5,14,17,19,28). For HCMV, the importance of intracellular transport of virus particles was underscored by the finding that cell tropism variants of HCMV are discriminated by the straindependent efficiency of nuclear translocation (25). By nature, translocation is a dynamic process, which can be analyzed only insufficiently by biochemical analyses of lysates of infected cells or by immunodetection of particles in fixed-cell preparations. At least the dynamic aspects of interactions between virus particles and target cells are best studied in live-imaging approaches that have been enabled by the development of green fluorescent virus variants. Recombinant fluor...