Adenovirus packaging cell lines for growth of an E1A/E1B deficient adenovirus that is substantially free of replication competent adenovirus (RCA) are provided. Methods for producing adenovirus substantially free of RCA are also provided, wherein the adenovirus is grown in a cell line containing coding sequences for adenovirus E1A and E1B, which are operably linked to promoters that lack polynucleotide sequences sharing substantial sequence identity with the native adenovirus E1A and E1B promoters.