We have developed a hybrid vector that combines the high transduction efficiency of a gene-deleted adenoviral vector and the integration machinery of the bacteriophage-derived integrase PhiC31 for stable transduction and limited integration sites. We based our system on a two-vector system in which the transgene expression cassette is circularized from a helper-dependent vector by Flp-mediated recombination, followed by PhiC31-mediated integration. Integration of the transgene expression cassette from the adenoviral vector resulted in 5-fold higher transgene expression levels in the active PhiC31 group compared to the control group, which received a mutated and inactive version of PhiC31. We confirmed transgene integration into the previously described mpsL1 hot spot of integration by polymerase chain reaction analyses of DNA isolated from mouse livers. In addition, we cloned 40 integration sites. The hot spot mpsL1 was detected only once, and 44% of all integration events were found to be present in gene regions. With further optimization, this system represents a new tool for gene therapy protocols that may offer an alternative to gene therapy approaches based on random integrating viral vectors.