Replication-incompetent adenovirus is conventionally produced by cells that supply replication-enabling proteins from viral sequences present in trans. As an alternative means of recombinant adenovirus production, replication-enabling E1A sequences were cotransduced into human prostate carcinoma cells infected with an E1A-deleted adenovirus containing a luciferase expression cassette. The replication-enabling plasmid was cotransduced by ionic linkage to the recombinant adenovirus exterior. Cells cotransduced with the replication-enabling plasmid made new adenovirus with titers up to 8 × 106 in the supernatants 72–120 hr after transduction. Like the parent virus, the virus present in the cotransduced supernatants and lysates was capable of transferring luciferase activity to new cells. The virus present in the cotransduced cell supernatants was amplified and shown to be identical to the parent virus by genomic analysis. It was concluded that simultaneous addition of a replication-defective adenovirus and a replication-enabling gene sequence in a trans configuration converts some of the cotransduced cells into recombinant adenovirus-producing cells.