Adenoviral vectors have been used in gene therapy and for vaccination. The major concerns with using adenoviral vectors are the pathogenic potential of the virus backbone and the generation of replication-competent adenovirus that may replicate in an uncontrolled manner, especially in immunocompromised patients. It is important to develop new vectors that are safer for clinical trials while maintaining high titer and efficient transduction. A new adenovirus vector production system was developed, which includes several vector backbone plasmids deleted for E2a and a new cell line expressing both E1 and E2a. The new cell line with the tTA-inducible E2a expression cassette can significantly increase the titer of E1/E2a-deleted vectors by four to five orders of magnitude upon withdrawal of tetracycline. Furthermore, there is no sequence overlap between the vector and the cellular DNA within the E2a open reading frame and downstream, making the generation of virus with wild-type E2a through homologous recombination substantially less likely. The new vector system may improve the safety of vectors for vaccination and cancer therapy and may also provide safer backbones for further vector development, such as helper-dependent and hybrid vectors.