Gene therapy is a novel method under investigation for the treatment of genetic, metabolic and neurologic diseases, cancer and AIDS. The primary goal of gene therapy is to deliver a specific gene to a pre-determined target cell, and to direct expression of such a gene in a manner which will result in a therapeutic effect. Retroviral vectors have the ability to integrate in the host cell DNA irreversibly and therefore, are suitable vectors for permanent genetic modification of cells. Retrovirus-mediated gene transfer has been limited, however, by the inability of onco-retroviruses to productively infect non-dividing cells. Lentiviruses are unique among retroviruses because of their ability to infect target cells independently of their proliferation status. This chapter presents an up-to-date description of available lentiviral vectors, including vector design, applications to disease treatment and safety considerations. In addition, general aspects of the biology of lentiviruses with relevance to vector development will be discussed.