Highly active antiretroviral therapy has greatly reduced the morbidity and mortality from human immunodeficiency virus (HIV) infection, but AIDS continues to be a serious health problem worldwide. Despite enormous efforts to develop a vaccine, there is still no cure, and alternative approaches including gene therapy should be explored. In this study we developed and compared combinatorial foamy virus (FV) anti-HIV vectors that also express a mutant methylguanine methyltransferase (MGMTP140K) transgene to increase the percentage of gene-modified cells after transplantation. These FV vectors inhibit replication of HIV-1 and also the simian immunodeficiency virus/HIV-1 (SHIV) chimera that can be used in monkey AIDS gene therapy studies. We identified a combinatorial FV vector that expresses 3 anti-HIV transgenes and inhibits viral replication by over 4 logs in a viral challenge assay. This FV anti-HIV vector expresses an HIV fusion inhibitor and two short hairpin RNAs (shRNAs) targeted to HIV-1 tat and rev, and can be produced at high titer (3.8 x 10(7) transducing units ml(-1)) using improved helper plasmids suitable for clinical use. Using a competitive repopulation assay, we show that human CD34(+) cells transduced with this combinatorial FV vector efficiently engraft in a mouse xenotransplantation model, and that the percentage of transduced repopulating cells can be increased after transplantation.