The ability of adenovirus to induce cell transformation depends on the E1A and E1B-55K oncoproteins. While E1A functionally inactivates the retinoblastoma tumour suppressor, E1B-55K primarily interferes with the function of p53. In adenovirus transformed cells E1B-55K can directly affect p53-dependent transactivation. In virus-infected cells E1B-55K additionally cooperates with the viral E4orf6 protein to induce ubiquitin-dependent degradation of p53. Here we unravel a novel activity of E1B-55K by demonstrating that it drastically stimulates the post-translational modification of p53 by the ubiquitin-like SUMO modifier. Consistent with this finding the extent of p53 SUMOylation is highly elevated in adenovirus transformed cell lines. E1B-55K-mediated SUMOylation depends on the direct interaction of E1B-55K with p53 and additionally requires SUMO modification of E1B-55K. These data suggest that E1B-55K exploits both ubiquitin and ubiquitin-like systems to target host cell proteins and thus shed new light on the molecular mechanisms of E1B- 55K function. Moreover, the data expand the emerging concept of dual-specificity factors that act in both the SUMO and ubiquitin pathway and identify E1B-55K as the first viral protein that shares this dual activity.