PML nuclear bodies (PML NBs), also called ND10, are matrix-bound nuclear structures that have been implicated in a variety of functions, including DNA repair, transcriptional regulation, protein degradation, and tumor suppression. These domains are also known for their potential to mediate an intracellular defense mechanism against many virus types. This is likely why they are targeted and subsequently manipulated by numerous viral proteins. Paradoxically, the genomes of various DNA viruses become associated with PML NBs, and initial sites of viral transcription/replication centers are often juxtaposed to these domains. The question is why viruses start their transcription and replication next to their supposed antagonists. Here, we report that PML NBs are targeted by the adenoviral (Ad) transactivator protein E1A-13S. Alternatively spliced E1A isoforms (E1A-12S and E1A-13S) are the first proteins expressed upon Ad infection. E1A-13S is essential for activating viral transcription in the early phase of infection. Coimmunoprecipitation assays showed that E1A-13S preferentially interacts with only one (PML-II) of at least six nuclear human PML isoforms. Deletion mapping located the interaction site within E1A conserved region 3 (CR3), which was previously described as the transcription factor binding region of E1A-13S. Indeed, cooperation with PML-II enhanced E1A-mediated transcriptional activation, while deleting the SUMO-interacting motif (SIM) of PML proved even more effective. Our results suggest that in contrast to PML NB-associated antiviral defense, PML-II may help transactivate viral gene expression and therefore play a novel role in activating Ad transcription during the early viral life cycle.