The Epstein-Barr virus (EBV) EBNA-LP protein is important for EBV-mediated B-cell immortalization and is a potent gene-specific coactivator of the viral transcriptional activator, EBNA2. The mechanism(s) by which EBNA-LP functions as a coactivator remains an important question in the biology of EBV-induced B-cell immortalization. In this study, we found that EBNA-LP interacts with the promyelocytic leukemia nuclear body (PML NB)-associated protein Sp100 and displaces Sp100 and heterochromatin protein 1alpha (HP1alpha) from PML NBs. Interaction between EBNA-LP and Sp100 was mediated through conserved region 3 in EBNA-LP and the PML NB targeting domain in Sp100. Overexpression of Sp100 lacking the N-terminal PML NB targeting domain, but not a mutant form of Sp100 lacking the HP1alpha interaction domain, was sufficient to coactivate EBNA2 in a gene-specific manner independent of EBNA-LP. These findings suggest that Sp100 is a major mediator of EBNA-LP coactivation. These studies indicate that modulation of PML NB-associated proteins may be important for establishment of latent viral infections, and also identify a convenient model system to investigate the functions of Sp100.