Human cytomegalovirus (HCMV) is the major viral cause of birth defects and a serious problem for immunocompromised individuals. Here we show that infection of cells with HCMV during the S-phase of the cell cycle results in two specific chromosome 1 breaks at positions 1q42 and 1q21. We demonstrate that purified virions, and not infected cell supernatant alone, are responsible for the damage. In addition, we show that the specific breaks occur when different sources of fibroblasts and strains of HCMV are used. Incubation of the virus with neutralizing antibody prevents the induction of breaks. However, UV-inactivated virus is as efficient as untreated virus in inducing specific damage to chromosome 1. Thus, there is a requirement for viral adsorption/penetration, but not new viral gene expression. This HCMV-mediated induction of site-specific damage in actively dividing cells may provide clues for the development of neurological defects in the congenitally infected infant.