Recombinant lentiviral vectors derived from human immunodeficiency virus-1 are promising vectors for gene therapy applications. This is due to their ability to transduce nondividing cells, their genomic stability, their relatively large genomic capacity, as well as their ability to stably maintain long term transgene expression. Recent studies have uncovered that the central polypurine tract (cPPT) plays an important role in increasing lentivirus-mediated gene transfer efficiency. The cPPT participates in lentiviral reverse transcription by initiating the synthesis of a downstream plus strand cDNA. Together with the upstream plus strand initiated at the 3' polypurine tract, it forms a central DNA flap that bears in its center a short plus strand overlap. A possible role in nuclear import enhancement of the viral preintegration complex has been suggested (Zennou, Cell, 2000), but the mechanism of cPPT function is still not fully elucidated. It has also been reported that the incorporation of cPPT in lentiviral vectors can increase particle infectivity (Follenzi, Nature Genetics, 2000). However, a systematic evaluation of the cPPT in complex lentiviral vectors has not been reported to date.