The rapid advancement of lentivirus-based gene transfer systems and their demonstrated utility in a variety of in vitro and in vivo settings has heightened the need for assays to evaluate the safety of these vectors prior to human clinical trials. Two major concerns relating to the use of lentivirus-based vectors in a clinical setting are the presence of contaminating replication-competent retroviruses in vector preparations and the efficiency of vector mobilization and spread by wild-type helper virus (rescue). This article describes an in vitro system to study the rescue of lentivirus-based vectors by wild-type HIV. We show that lentivirus-based vectors can be readily rescued from T cell lines and to a lesser extent from primary human lymphocytes by wildtype HIV, resulting in the spread of mobilized vector particles to previously untransduced cells. Furthermore, we show that vector mobilization can be prevented by antiretroviral drugs such as AZT. In contrast to recently published reports by Bukovsky et al. and An et al., the lentivirus vectors used in these studies had little or no effect on the replication and spread of HIV in transduced cells [Bukovsky et al. (1999). J. Virol. 73, 7087-7092; An et al. (1999). J. Virol. 73, 7671-7677]. Whereas vector spread is a significant concern for most gene therapy applications, in the context of gene therapy for HIV infection it may have beneficial effects.