The nuclear replication and retention functions of the Epstein-Barr virus (EBV) have been utilized here to maintain retroviral constructs episomally within human cell-based retroviral packaging lines. These hybrid EBV/retroviral constructs are capable of producing helper-free recombinant retrovirus as soon as 48 hr and for at least 30 days after transfection into 293T-based ecotropic and/or amphotropic retroviral packaging cells. Viral titers greater than 10(7) TU/ml were obtained after puromycin selection of transfected retroviral packaging cells. This episomal approach to retroviral production circumvents some limitations inherent in transient and chromosomally stable retroviral producer systems, affording reproducibly rapid, large-scale, stable, and high-titer retrovirus production.