Many applications of gene delivery require long-term transgene expression. In dividing cells, this result necessitates vector genome persistence, usually by integrating into cellular DNA. Since recombinant gene delivery vectors derived from tag-deleted, replication-incompetent simian virus-40 (SV40) provide for long-term transgene expression in resting and dividing cells, we tested whether such enduring transgene expression reflected integration into cellular genomes. Several lines of evidence suggested this likelihood. After transduction in vitro, continuously dividing cell lines and continuously stimulated primary cells uniformly showed transgene expression for many months. Mice whose livers were transduced in vivo, partially resected, and allowed to regenerate showed comparable levels of transgene expression in regenerated and preoperative livers. Thus, replicationincompetent SV40 vectors (rSV40) persist in vitro and in vivo despite extensive cell division. We tested the possibility that this persistence reflected integration directly. Southern blot analyses of genomic DNA from transduced 293 cells showed that vector genome incorporation into cell DNA happened within days of transduction. Episomal vector DNA was barely detectable 96 hours post-transduction. Inverted PCR, used to characterize vector integration points, showed vector DNA integrated randomly into the cell genome. The circular rSV40 genome opened at different points in each integrand. A significant proportion of the integrands did not contain the entire vector sequence, but rather only portions thereof. Quantitative Southern blot analysis showed approximately 3.05 transgene copies per cell. Therefore, recombinant SV40 gene delivery vectors integrate into the cellular DNA of both resting and dividing cells, and do so randomly and within days of transduction. This integration may explain long-term transgene expression.