Human herpesvirus 6 (HHV-6) is a T-lymphotrophic virus belongs to the genusRoseoloviruswithin the beta herpesvirus subfamily. The U20–U24 gene cluster is unique toRoseoloviruses; however, both their function and whether they are essential for virus growth is unknown. Recently, bacterial artificial chromosome (BAC) techniques have been used to investigate HHV-6A. This study describes generation of a virus genome lacking U21–U24 (HHV-6ABACΔU21–24) and shows that infectious virus particles can be reconstituted from this BAC DNA. Our data indicate that the HHV-6 U21–U24 gene cluster is dispensable for virus propagation.