Reverse transcription of the human immunodeficiency virus type I is characterized by the formation of a DNA flap at the center of the viral cDNA in between the central polypurine tract (cPPT) and the central termination sequence (CTS). The importance of the DNA flap for HIV-1 replication has been questioned, whereas its importance for lentiviral vector performance is well accepted. To investigate this controversy, we re-evaluated the importance of the DNA flap for HIV-1 replication. A flap negative HIV-1 virus showed a 10- to 100-fold replication defect in comparison with a WT strain. Further characterization of the DNA flap in the context of lentiviral vectors showed that mutations in the DNA-flap sequence did not affect the transduction efficiency. Finally, introduction of a second cPPT/CTS sequence resulted in the presence of two DNA flaps but no higher transduction efficiency. (c) 2006 Elsevier Inc. All rights reserved.