The nonstructural protein NS1 of the autonomous parvovirus minute virus of mice interferes with cell division and can cause cell death, depending on the cell transformation state. Upon infection, the synthesis of NS1 protein is massively initiated during S phase. In this article, we show that minute virus of mice-infected cells accumulate in this phase. To investigate the link between NS1 accumulation and S-phase arrest, we have used stably transfected cells in which NS1 expression is under the control of a glucocorticoid-inducible promoter (the long terminal repeat of mouse mammary tumor virus). NS1 expression interferes with cell DNA replication, and consequently, the cell cycle stops in S phase. NS1 expression also induces nicks in the cell chromatin, as detected by an in situ nick translation assay. The nicks are observed several hours before any cell cycle perturbation. As cell cycle arrest is a common consequence of DNA damage, we propose that NS1 exerts its cytostatic activity by inducing lesions in cell chromatin.