Early during infection, the herpes simplex regulatory protein ICP0 promotes the proteasome-dependent degradation of a number of cellular proteins and the loss of a number of SUMO-1-modified protein isoforms, including PML. Recently, ICP0 has been shown to induce the accumulation of conjugated ubiquitin and function as a ubiquitin E3 ligase. However, certain aspects of the biochemistry, cell biology and the links between SUMO-1 conjugation/deconjugation and protein degradation remain unclear. For example, it is not currently known whether SUMO-1 deconjugation is a prerequisite for ubiquitination or degradation and, if so, by what mechanism this may occur. To help address these questions, a SUMO-specific protease (SENP1) was cloned and its expression and localization in relation to ICP0 examined. A cell line was established which constitutively expresses SUMO-1 to facilitate studies of localization and biochemistry. SENP1 localized to the nucleus mainly in discrete subdomains, a subset of which co-localized with the PML bodies. Both ICP0 and SENP1 protease promoted the loss of SUMO-1 from the nucleus, observed both for the endogenous species and the cell line expressing the epitope-tagged SUMO-1. The tagged SUMO-1 was recruited into high molecular mass conjugates in the cell line, and expression of SENP1 promoted loss of these species, including the modified species of PML. Finally, in co-transfection experiments ICP0 promoted the recruitment of SENP1 to nuclear domains, a result which was also observed early during infection. The significance of these findings is discussed in relation to the function of ICP0.