Deletion of viral functions that can be complemented by the specific phenotype of tumor cells is a common strategy to design oncolytic viruses. For example, enhanced mRNA cytoplasmic export in tumor cells phenocopies the adenovirus E1B-55K function and renders mutants of this protein tumor selective. Also, an activated RB pathway complements specific E1A functions that can be deleted to produce oncolytic viruses. In this paper we demonstrate that an adenoviral mutant deleted in virus-associated I (VAI) and VAII RNAs (Ad-VAdel) has oncotropism characterized by 100-fold replication deficiency compared with wild-type adenovirus in normal cells and an unaffected ability to replicate and kill different types of tumor cells. This mutant also displays active antitumoral activity in vivo. In contrast, this oncotropism is less evident in a mutant expressing an inactive form of VAI (Adsub719) because VAII RNA expression is upregulated. The mRNA translation promoted by VA RNA genes can be phenocopied in tumor cells with the activation of signal transduction pathways, such as the Ras pathway.