Nonintegrating gene delivery vectors have an improved safety profile compared with integrating vectors, but transgene retention is problematic as nonreplicating episomes are progressively and rapidly diluted out through cell division. We have developed an integration-deficient lentiviral vector (IDLV) system generating mitotically stable episomes capable of long-term transgene expression. We found that a transient cell cycle arrest at the time of transduction with IDLVs resulted in 13-45% of Chinese hamster ovary (CHO) cells expressing the transgene for over 100 cell generations in the absence of selection. The use of a scaffold/matrix attachment region did not result in improved episomal retention in this system, and episomes did not form after transduction with adeno-associated viral or minicircle vectors under the same conditions. Investigations into the episomal status of the vector genome using (1) linear amplification-mediated polymerase chain reaction followed by deep sequencing of vector-genome junctions, (2) Southern blotting, and (3) fluorescent in situ hybridization strongly suggest that the vector is not integrated in the vast majority of cells. In conclusion, we have developed an IDLV procedure generating mitotically stable episomes capable of long-term transgene expression. The application of this approach to stem cell populations could significantly improve the safety profile of a range of stem and progenitor cell gene therapies.