Recent advances in adeno-associated virus (AAV) vector production have eliminated the need for adenovirus infection by transfection of plasmids encoding the adenovirus E2A, E4orf6, and VA RNA transcription units. We report here the generation of significantly higher AAV vector titers with transfection of the single adenovirus gene, E4orf6, when used in conjunction with the split AAV packaging plasmids MTrep and CMVcap. Transduction in a murine lung model with these higher titer vector stocks was greater than that observed with traditional preparation methods. The generation of higher titer AAV vector stocks with fewer adenovirus gene products and free of replication-competent AAV will enhance the potential for AAV in clinical applications.