During reverse transcription of the human immunodeficiency virus type I (HIV-1), a 90- or 99-nucleoticle long DNA flap is formed at the centre of the viral cDNA. The presence of a central DNA flap in lentiviral vectors improves transduction efficiency significantly. We analysed the stimulation of lentiviral vector transduction by a DNA flap present at ectopic positions in the viral cDNA. A HIV-1 vector containing the cPPT/CTS fragment immediately downstream of the 5'-LTR performed as well as the wildtype cPPT-vector. Cloning of the cPPT/CTS fragment in front of the 3'-LTR resulted as well in a vector with higher transduction efficiency than a vector without central flap. These results demonstrate that the position of the DNA flap is not essential for its function in the context of HIV-1-derived lentiviral vectors. This may have consequences for vector design and our understanding of the functioning of the HIV-1 DNA flap. (C) 2005 Elsevier Inc. All rights reserved.