Cell lines stably expressing rep/cap are important tools for studying adeno-associated virus (AAV) biology and producing AAV vectors. Several rep/cap cell lines have been isolated, each of which is based on HeLa cells. Infection of these cell lines with adenovirus for production of AAV vector is associated with substantial amplification of the rep/cap gene. Concerns over the presence of human papilloma viral (HPV) sequences in HeLa cells may limit use of such lines for production of clinical-grade vectors. Here we describe a non-HeLa-derived rep/cap cell line called K209, generated by stable transfection of A549 cells with a plasmid construct containing the P5 rep/cap cassette from AAV2. Infection of K209 cells with adenovirus leads to a 1000-fold amplification of the rep/cap gene with high-yield production of AAV vectors. The multiplicity of infection (MOI) of adenovirus that led to maximum amplification of the rep/cap gene and high-level production of AAV is 10 times higher in the HeLa-based cell line than that required in K209 cells. Our data suggest that papilloma-derived gene products present in HeLa cells are not required for high-yield production of AAV vectors.