Numerous viral vectors have been developed for the delivery of transgenes to specific target cells. For persistent transgene expression, vectors based on retroviruses are attractive delivery vehicles because of their ability to stably integrate their DNA into the host cell genome. Initially, vectors based on simple retroviruses were the vector of choice for such applications. However, these vectors can only transduce actively dividing cells. Therefore, much interest has turned to retroviral vectors based on the lentivirus genus because of their ability to transduce both dividing and non-dividing cells. The best characterized lentiviral vectors are derived from the human immunodeficiency virus type 1 (HIV-1). This chapter describes the basic features of the HIV-1 replication cycle and the many improvements reported for the lentiviral vector systems to increase the safety and efficiency. We also provide practical information on the production of HIV-1 derived lentiviral vectors, the cell transduction protocol and a method to determine the transduction titers of a lentiviral vector.