ICP0 is a multifunctional herpes simplex virus protein known primarily as a promiscuous transactivator. In the course of productive infection, it is localized during the first 5–7 h in the nucleus and later in the cytoplasm. In the nucleus, its primary activities are to suppress the silencing of viral DNA by host proteins, activate cdk4 through recruitment of cyclin D3 to the sites of formation of replication compartments, and degrade several cellular proteins including PML and Sp100, key components of the ND10 nuclear bodies. ICP0 is not translocated to the cytoplasm in cells infected with mutants incapable of performing these tasks. We report the unexpected finding that ICP0 is cleaved into several discrete polypeptides by a proteasome-independent process. The products of this cleavage accumulate in cells infected with ICP0 mutants incapable of degrading PML and therefore are retained in the nucleus. In the second step, the products of the initial cleavage of wild-type virus-infected cells are themselves subject to proteasome-dependent degradation. The average half life of intact ICP0 during the nuclear phase is approximately 1 h. The proteasome-independent cleavage products are no longer detected at late times corresponding to the cytoplasmic phase of ICP0. The results are consistent with the hypothesis that the cleavage products of ICP0 function in topologically distinct domains during its nuclear phase.