Herpes simplex virus type 1 (HSV-1) replication involves the expression of its genes in a interdependent and well ordered cascade. The immediate early (IE) genes are expressed first on viral DNA entry into the nucleus and do not require expression of any other viral functions. The IE gene product, ICP0, plays an important role in enhancing early and late gene expression and is required for virus reactivation from latency in peripheral nerves. ICP0 is also toxic for dividing cells primarily through its ability to induce cell cycle arrest at G1 and S in synchronized cells and G2/M arrest in nonsynchronized cells ( ). The mechanism of cell cycle arrest is unknown. In this report, we confirm that mutant viruses that only express ICP0 cause G2/M arrest. Following infection, ICP0 induces ATM phosphorylation at tyrosine 1981 and CHK2 phosphorylation at tyrosine 68, P53 phosphorylated in serine 15 and 20. These events lead to G2 arrest. Chk2-/- cells can not cause G2/M arrest after virus infection but rather results in apoptosis. Together these results provide a mechanism through which ICP0 arrests cell division an event important to efficient virus growth.