Despite the benefit of achieving stable transgene expression with retroviral-based vectors, adverse effects associated with the administration of these vectors include the potential for insertional mutagenesis. For gene transfer applications in which integration is not required, we propose that HIV-1 based lentiviral vectors defective for vector DNA integration will allow for clinically relevant levels of transgene expression. To this end, human 293 cells were transduced with either wild-type (integration-competent) or mutant (integration-deficient) GFP-expressing vectors. The integration-deficient vectors included one integrase (IN) deletion mutant (|[Delta]|174-288) and two class I integrase missense (D116N and E152V) mutants. IN/|[Delta]|174-288 was generated by deletion of the IN c-terminal domain and about 20% of the core domain, while IN/D116N and IN/E152V were generated by the incorporation of missense mutations within the active site of the IN core domain at conserved acidic residues. Physical titers of each vector preparation were determined upon measurement of p24 gag capsid protein production, so that the amount of vector particles could be normalized for each construct. Flow cytometric analysis was performed to assess transgene expression from vector transduced 293 cells up to 28 days post-transduction. At most, only about 2% of 293 cells express GFP upon transduction with IN/|[Delta]|174-288, suggesting that this deletion may function as a class II integrase mutation and affect other viral life cycle activities such as gag/pol processing. At an MOI of about five, nearly 30% of IN/D116N and IN/E152V transduced 293 cells expressed GFP at 48 hours post-transduction, compared to about 90% for vectors containing wild-type integrase. When assessing the mean fluorescence, the amount of GFP expression achieved is reduced about seven-fold relative to wild-type vector transduced 293 cells. Cell cycle status may influence GFP expression in IN/D116N transduced 293 cells. Relative to cycling 293 cells, a two-fold increase in the percentage of GFP expressing cells is observed in G1 and G2 arrested 293 cells, and mean fluorescence of GFP expression is increased two-fold in G1 arrested 293 cells. A reduction in GFP expression may reflect a lower level of transgene expression achieved from presumed unintegrated vector DNA in IN/D116N and IN/E152V transduced 293 cells. To assess the state of vector DNA integration, an Alu-based real-time quantitative PCR assay for integration was performed. Using this assay, relative to wild-type vector transduced 293 cells, no integration events could be detected in IN/D116N and IN/E152V transduced 293 cells. At 28 days post-transduction, about 2000 copies of integrated vector DNA per ng of cellular DNA were detected in wild-type vector transduced 293 cells. In characterizing the expression and integration status of vector DNA in integrase mutant vector transduced 293 cells, the above results suggests that the transient nature of transgene expression is these cells occurs as a result of expression from predominantly unintegrated vector DNA and is influenced by the cell cycle status.