Conditionally replicating adenoviruses are developing as a complement to traditional cancer therapies. Ad[I/PPT-E1A] is an E1B/E3-deleted virus that replicates exclusively in prostate cells, since the expression of E1A is controlled by the recombinant 1.4 kb prostate-specific PPT promoter. The transcriptional integrity of PPT is maintained by the 3.0 kb mouse H19 insulator that was introduced directly upstream of the PPT sequence. In order to increase the cloning capacity to be able to reintroduce E3 sequences in the 35.7 kb Ad[I/PPT-E1A] genome, various shorter insulators were examined in a luciferase reporter gene assay. It was found that the 1.6 kb core H19 insulator (i) improves the activity of PPT, compared to the 3.0 kb full-length insulator, while still maintaining prostate cell specificity and releasing 1.4 kb of space for insertion of additional sequences. To improve the ability of the virus to efficiently lyse infected cells and persist in vivo, we inserted the adenovirus death protein (ADP) or the full-length adenovirus E3 region. The oncolytic activity of PPT-E1A-based viruses was studied using MTS, crystal violet and replication assays. The virus with the reintroduced full-length E3-region (Ad[i/PPT-E1A, E3]) showed the highest cytopathic effects in vitro. Furthermore, this virus suppressed the growth of aggressively growing prostate tumors in vivo. Therefore, we conclude that Ad[i/PPT-E1A, E3] is a prostate-specific oncolytic adenovirus with a high potential for treating localized prostate cancer.