Viral vectors based on recombinant adenoviruses (rAd) are used extensively as gene delivery systems for investigations of gene function and gene therapy applications. In both cases, the desire is for efficient expression of a transgene without substantial toxicity to the target cell. The first generation of rAd vectors were deleted of early region E1 and sometimes additionally early region E3. Evaluation of these vectors in preclinical animal models revealed transient gene expression, accompanied by cytotoxicity and immune responses. Expression of remaining viral genes, even in the absence of the E1 region, contributes to these effects and therefore it is important to have an appreciation of the functions of these viral genes. In particular, the early region E4 has been shown to affect transgene persistence, vector toxicity and immunogenicity. Proteins from the E4 genes can modulate transcription, the cell-cycle, cell signaling and DNA repair. In addition, some of these proteins also cause oncogenic transformation. Therefore interactions of these viral genes with key cell regulators should be taken into account when engineering rAd vectors. This review will summarize our knowledge of E4 functions and the implications of recent findings on the development of rAd vectors.