The ultrastructural replication cycle of human herpesvirus 6A and 6B, both T-lymphotropic viruses, with tropism for the central nervous system, was compared by electron microscopy in the same cells, that is, in the T-lymphoblastoid cell line SupT-1 and in human astrocytes. Both HHV-6A and HHV-6B replicated efficiently in SupT-1 and formed viral particles. The tegument is the least characterized structure of the herpesviral particle and both variants were able to form intranuclear membrane compartments called tegusomes in SupT-1 where tegumentation occurred. Also, tegumentation occurred in HHV-6A infected cells in the nucleoplasm without the presence of a tegusome. This suggests that there is more than one possible route of tegumentation. Differences in the replication cycles between HHV-6A and HHV-6B were also observed in the cytoplasm. One such difference was that prominent annulate lamellae were only found in the cytoplasm of HHV-6A infected cells. In astrocytes a successful formation of viral particles was only seen with the HHV-6A variant. The HHV-6A virus life cycle in astrocytes resembled the life cycle in the T-cell line SupT-1, except that no annulate lamellae were found. Complete viral particles were found extracellularly around the astrocytes and the supernatant of infected astrocytes were able to re-infect SupT-1 cells. This suggests that HHV-6A infection in astrocytes can generate complete, viable, and infectious viral particles. The HHV-6 variants behave differently in the same type of cells and have different tropisms for astrocytes, supporting the notion that the variants might induce different diseases. J. Med. Virol. 78:1542–1553, 2006. © 2006 Wiley-Liss, Inc.