Preparations for a phase I trial of ex vivo, anti-HIV ribozyme gene therapy have included optimization of transduction and expansion of CD4+ lymphocytes from HIV-1 infected donors, using reagents suitable for production of cell products for human infusion. We also determined whether transduction by the ribozyme vector would inhibit replication and spread of endogenous HIV-1, and result in preferential survival of ribozyme-transduced CD4+ cells during lymphocyte expansion. Transduction efficiency, as estimated by DNA quantitative competitive (QC)-PCR, was similar for both control (LNL6) and ribozyme expressing (MJT) murine retroviral vectors (approximately 20%.) In the absence of antiviral agents, cells transduced with MJT exhibited three-fold greater numbers of CD4+ cells 2 weeks after transduction than did LNL6 transduced cells. In addition, viral replication was delayed 2-3 weeks in MJT transduced cultures. Both transduced cell populations expanded by 2-3 logs within 2 weeks. The clinical protocol involves infusion of both ribozyme and control vector transduced cells, making identification of agents capable of suppressing replication and spread of endogenous virus during ex vivo expansion necessary. The combination of nevirapine (100 nM) and CD4-PE40 (100 nM) completely suppressed endogenous virus replication in cultures transduced with either vector. At reduced concentrations of nevirapine, virus replication was suppressed only in MJT transduced cells.