Retroviral-mediated gene transfer into bone marrow cells is extensively used in gene therapy protocols. Cytokines are needed for stimulation, to achieve a high rate of gene transfer. However, the stromal cell compartment of bone marrow is characterized by rapid proliferation even without cytokines. In this study, human bone marrow stromal cells were isolated and subsequently infected with recombinant retrovirus in a cell-free supernatant. The LN retroviral vector used in this study carries the bacterial neomycin phosphotransferase gene (neoR). Transduction efficiency was significantly enhanced by repeated cycles of infection, with a maximum of 91% transduced cells by four rounds of infection. Presence of the neoR-gene was detected by PCR from all stromal cells selected by G418. After culture in vitro for 3 months, cells were still positive for PCR amplification of the neoR-gene. Transduced stromal cells were also injected into SCID mice to study their homing and survival ability in vivo.